RECOMBINANT SEROTYPE 5 (Ad5) ADENOVIRAL VECTORS

ABSTRACT

The invention relates to oncolytic adenovirus vectors and their uses in cancer therapy. The adenovirus vectors according to the invention have superior safety properties and have effective therapeutic activity. A production method for the inventive adenoviruses is also disclosed. The adenovirus vectors are useful in cancer therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.:14/359,141, filed May 19, 2014 which was a 35 U.S.C. §371 National PhaseEntry Application of International Application No. PCT/FI12/51162, filedNov. 23, 2012. PCT/FI12/51162 claims benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application Ser. No.: 61/563,634, filed Nov. 25, 2011,and Finnish Application No. 20116181, filed Nov. 25, 2011. The contentsof the aforementioned applications are incorporated herein by referencein their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 25, 2015, isnamed ONCT-002DO1US ST25.txt and is 401,366 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of medicine. Specifically,the present invention relates to novel strategies to construct oncolyticadenoviral vectors for cancer therapy allowing safe and efficienttreatment.

BACKGROUND OF THE INVENTION

Typically, cancer is treated with conventional treatment regimens suchas surgery, hormonal therapies, chemotherapies, radiotherapies and/orother therapies. However, in many cases cancers which often arecharacterized by an advanced stage cannot be cured with presenttherapies. Despite progress in conventional cancer treatment regimens,metastatic disease essentially remains incurable and new treatmentalternatives are desperately needed.

Virotherapy is a relatively novel treatment approach, which harnessesthe natural ability of some viruses to kill the cells in which theyproliferate and the ability to spread to neighboring cells, therebyamplifying the therapeutic effect of the initial input dose.Requirements of optimal viral vectors include an efficient capability tofind specific target cells and express the viral genome in the targetcells. Furthermore, therapeutically optimal vectors have to stay activein the target tissues or cells long enough to exert their therapeuticefficacy while causing minimal effects in normal cells. There has beensome progress in developing these beneficial properties of therapeuticviral vectors during the last decades and, for example, retroviral,adenoviral and adeno-associated viral vectors have been widely studiedin biomedicine.

Contrary to the viral gene therapy approach, in which foreign geneticmaterial is introduced in cells to correct genetic defects, oncolyticvirotherapy takes advantage of the similarities between cellularmechanisms of carcinogenesis and DNA virus replication to direct thecell lysing activity of an oncolytic virus to tumor. In virotherapy thecancer cell transduction and viral replication are carefully controlledby genetic engineering of the viral genome to gain effective and safetumor eradication. In other words, the use of replicating, oncolyticviruses for cancer treatment necessitates introduction of variousgenetic modifications to the viral genome, thereby restrainingreplication exclusively to tumor cells and eventually obtainingselective eradication of the tumor without side effects to healthytissue.

Upon infection, adenoviruses need to induce a cell cycle S-phase-likestate in order to transcribe and replicate the viral genome. E1A is thefirst viral protein to be expressed in a transduced cell and it canactivate transcription of other early viral genes by interactions withcellular check point proteins. Importantly, E1A expression results inthe activation of the Eta promoter and the E2 region transcription,leading to the expression of adenoviral replication machinery (Berk1986, Annu Rev Genet 20: 45-79).

Specific deletions on adenoviral key regulatory genes have been utilizedto create dysfunctional proteins or the lack of their expression thatleads to dependence on a specific genetic feature present in targetcells. Partial deletions of E1A result in restricted replication innormal cells but allow replication in target cells, such as cancercells. Conditionally replicating viruses featuring a 24 base pairdeletion in the CR2 (constant region 2) have been created and shown tobe potent and selective in the treatment of glioma and breast cancerxenografts (Fueyo et al. 2000, Oncogene 19:2-12; Heise et al. 2000, NatMed 6:1134-9). Their cancer specificity results from the inability ofdysfunctional E1A to release E2F1 transcription factor, which leads tothe requirement of free E2F 1. E2F 1 is abundant in cancer cells, wherethe pRb pathway is most often disrupted (Hanahan and Weinberg 2000, Cell100:57-70).

Most clinical trials have been performed with early generationadenoviruses based on adenovirus 5 (Ad5). The anti-tumor effect ofoncolytic adenoviruses depends on their capacity for gene delivery.Unfortunately, most tumors have low expression of the main Ad5 receptor,coxsackie-adenovirus receptor (CAR).

Currently most oncolytic viruses in clinical use are highly attenuatedin terms of replication due to several deletions in critical viralgenes. These viruses have shown excellent safety record, but theantitumor efficacy has been limited. However, clinical and preclinicalresults show that treatment with unarmed oncolytic viruses is notimmunostimulatory enough to result in sustained anti-tumoral therapeuticimmune responses. In this regard, oncolytic viruses have been armed tobe more immunostimulatory. Virally infected cells are superior atdelivery of nonviral antigen (i.e. tumor antigen) for cross-presentation(Schulz et al. 2005, Nature 433:887-92), and virally induced cell deathwould be expected to enhance the availability of tumor-associatedantigens for uptake by dendritic cells (DCs) (Moehler et al. 2005, HumGene Ther 16:996-1005) and subsequently enhance stimulation of cytotoxicT-cells. Furthermore, viral infection may alter the balance of cytokineproduction from the tumor, and subsequently affect the nature of theimmune reaction to the tumor, that is, by counteracting theimmunosuppressive nature of the tumor microenvironment (Prestwich et al.2008, Expert Rev Anticancer Ther 8:1581-8). Most importantly, virusescan be engineered to express highly immunogenic proteins such asgranulocyte-macrophage colony-stimulating factor (GM-CSF). Whenimmunogenic proteins are expressed within tumor cells, they are potentstimulators of specific and long-lasting antitumor immunity.Introduction of immunotherapeutic genes into tumor cells and,furthermore, their translation into proteins, leads to the activation ofthe immune response and to more efficient destruction of tumor cells.The most relevant immune cells in this regard are natural killer cells(NK) and cytotoxic CD8+ T-cells.

Adenoviral Vectors

Adenoviruses are non-enveloped viruses 70-90 nm in diameter with anicosahedral capsid. Their genome is linear, double stranded DNA varyingbetween 25-45 kilobases in size with inverted terminal repeats (ITRs) atboth termini and a terminal protein attached to the 5′ ends (Russell2000, J gen Virol 90:1-20).

The icosahedral capsid is formed by three major proteins, of which thehexon trimers are most abundant (Nemerow et al. 2009, Virology384:380-8). Each of the twelve vertices of the capsid also contains apentameric protein, a penton base that is covalently attached to thefiber. The fiber is a trimeric protein that protrudes from the pentonbase and is a knobbed rod-like structure. Other viral proteins Ma, IVa2,VI, VIII and IX are also associated with the viral capsid. The proteinsVII, small peptide mu and a terminal protein (TP) are associated withDNA. Protein V provides a structural link to the capsid via protein VI.

All human adenoviruses have similarities in their fiber architecture.Each has an N-terminal tail, a shaft with repeating sequences, and aC-terminal knob domain with a globular structure. The knob domain isprincipally responsible for binding the target cellular receptor and itsglobular structure presents a large surface for lateral and apicalbinding. The fiber proteins of adenoviruses from different subgroupsmost distinctively differ in length and ability to bend.

The fiber participates in attachment of the virus to the target cell.First, the knob domain of the fiber protein binds to the receptor of thetarget cell, secondly, the virus interacts with an integrin molecule,and thirdly, the virus is endocytosed into the target cell. Next, theviral genome is transported from endosomes into the nucleus and thereplication of the viral genome can begin (Russell W. C. 2000, J GeneralVirol 81, 2573-2604).

Adenoviruses are dependent on the cellular machinery to replicate theviral genome. They can infect quiescent cells and induce them into acell cycle S-phase-like state enabling viral DNA replication. Theadenoviral genome can be divided into immediate early (E1A), early (E1B,E2, E3, E4), intermediate (IX, Iva), and late (L1-L5) genes (Russell2000).

Adenoviral transcription can be described as a two-phase-event, earlyand late, characterized by the expression of different viral genes andseparated by the onset of viral DNA replication (Russell 2000, J genVirol 90:1-20). The first transcription unit to be expressed is the E1A.The E1A proteins stimulate the transcription of other early genes andmodulate the expression of cellular genes involved in the transitioninto S-phase, making the cell more susceptible to viral DNA replication(Berk 1986, Annu Rev Genet 20: 45-79). The E1B proteins suppress celldeath elicited in response to unregulated cell proliferation signals,including those mediated by E1A (Moran 1993, FASEB J 7:880-5). The E2gene products provide the replication machinery for viral gene products.

E3 gene products are not essential for virus replication in vitro, butare dedicated to the control of various host immune responses. E3-gp19Kinhibits the transport of the class 1 major histocompatibility complex(MHC) from the endoplasmic reticulum (ER) to the plasma membrane,thereby preventing the presentation of peptides to T lymphocytes by MHC(Rawle et al. 1989, J Immunol 143:2031-7). Other E3 proteins inhibitapoptosis elicited by various cellular proteins such as the tumornecrosis factor α (TNFα) (Wold 1993, J Cell Biochem 53:329-35). As anexception, E3 derived adenoviral death protein (ADP) functions late inthe viral cycle to promote cell death, presumably to aid in the releaseof the virus after all the replicative functions have been completed. E4gene products have been implicated in many events that occur as the lateprogram begins. E4 proteins augment viral DNA synthesis and messengerRNA (mRNA) transport, late viral gene expression, shutoff of hostprotein synthesis, and production of progeny virions. The late genetranscription leads to the production of viral structural components andthe encapsidation and maturation of the viral particles in the nucleus.

More than 50 different serotypes of adenoviruses have been found inhumans. Serotypes are classified into six subgroups A-F and differentserotypes are known to be associated with different conditions i.e.respiratory diseases, conjunctivitis and gastroenteritis. Adenovirusserotype 5 (Ad5) is known to cause respiratory diseases and it is themost common serotype studied in the field of gene therapy. In the firstAd5 vectors E1 and/or E3 regions were deleted enabling insertion offoreign DNA to the vectors (Danthinne and Imperiale 2000, Gene Ther7:1707-14). Furthermore, deletions of other regions as well as furthermutations have provided extra properties to viral vectors. Indeed,various modifications of adenoviruses have been suggested for achievingefficient anti-tumor effects.

EP1377671 B1 (Cell Genesys, Inc.) and application US2003/0104625 A1(Cheng C. et al.) describe an oncolytic adenoviral vector encoding animmunotherapeutic protein granulocyte-macrophage colony-stimulatingfactor (GM-CSF).

EP1767642 A1 (Chengdu Kanghong Biotechnologies Co., Ltd.) disclosesoncolytic adenoviral vectors having improved effects on human immuneresponse.

WO2010072900 discloses oncolytic adenoviral vectors having a modifiedviral genome and an immunostimulatory GM-CSF.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel oncolyticadenoviruses for cancer therapy and to solve problems relating toconventional cancer therapy and manufacture of therapeutically effectiveand safe viral therapies for cancer.

Modifications in the E1-Region

Pan-cancer promoters target hallmark cancer pathways making them broadlyapplicable for targeting approaches in various cancer types. Examples ofsuch promoters are E2F-1, the human telomerase reverse transcriptase(hTERT) promoter, and the multidrug resistance promoter (Mdrl). In theviruses of the present application the native E1A promoter has beenreplaced by a human E2F-1 promoter to control the expression of E1A andsubsequent viral replication. E2F transcription factors regulate theexpression of a diverse set of genes involved in key cellular events bybinding to their promoters (Johnson and Schneider-Broussard 1998, FrontBiosci 3:447-8). E2F transcription factors also activate their ownpromoters. In a resting cell E2F transcription factors are bound in acomplex with retinoblastoma (pRb) protein. The pRb/E2F-1 complexinactivates the E2F-1 promoter and E2F-1 promoter activation requiresfree E2F-1 transcription factor. The pRb pathway is disrupted in nearlyall human cancers, resulting in abundant free E2F-1 in cancer cells.This creates a broad target spectrum for E2F-1 promoter usage inreplication control of oncolytic viruses in cancer treatment.

The rationale behind the use of a 24 base pair deletion in the E1A genefor restricting viral replication to cancer cells is similar to the useof E2F-1 promoter. The adenoviral E1A protein was originally describedas a pRb binding protein capable of inducing DNA replication inquiescent normal cells (Ruley 1983, Nature 304:602-6). One of the keyfunctions of E1A protein is to disrupt the pRb-E2F interactions, therebyreleasing E2F transcription factors to activate the E2F responsivepromoters and transcription of the genes they control, such asadenoviral E2A (Raychaudhuri et al. 1991, Genes Dev 5:1200-11). Theconserved region 2 (CR2) in E1A protein forms a strong interaction withthe pocket binding domain of pRb and CR1 mediates the actual disruptionof the E2F binding of pRb (Fattaey et al. 1993, Mol Cell Biol13:7267-77). Conditionally replicating viruses featuring a 24 base pairdeletion in the CR2 were created and shown to be potent and selective inthe treatment of glioma and breast cancer xenografts (Fueyo et al. 2000,Oncogene 19:2-12; Heise et al. 2000, Nat Med 6:1134-9). Their cancerspecificity results from the inability of dysfunctional E1A to releaseE2F1 transcription factor, which leads to the requirement of free E2F1,similarly as when controlling the E1A expression with E2F-1 promoter.

However, one critical aspect has been neglected in previous virusesfeaturing the E2F-1 promoter. Since the promoter has E2F-1 bindingsites, and is therefore effectively self-activated, even minute amountsof free E2F-1 (as found in normal cells) would lead to activation of thepromoter, for release of more E2F-1 as a result of E1A binding to Rb.Eventually, this vicious loop leads to replication of such viruses innormal cells. Therefore, selectivity of the promoter can only beretained by inactivating binding of Rb by E1A, as described in thispatent.

Modifications in the E3 Region

Adenoviruses are immunogenic viruses (Cerullo et al. 2007, Mol Ther15:378-85), and since it seems that the immune response is a majordeterminant of the antitumor effect of oncolytic viruses (Tuve et al.2009, Vaccine 27:4225-39), they have a great potential for cancertherapy utilities. Based on the “danger signal” paradigm (Matzinger1994, Annu Rev Immunol 12:991-1045), the presence of oncolytic viruseswithin a tumor can act as a danger signal for the immune system.Further, tumor associated antigens (TAAs) are self-derived moleculesthat are converted immunogenic due to various genetic alterations and,as such, can be viewed as a second danger signal when released fromcells undergoing abnormal death by viral oncolysis. The immunity relatedto adenoviral replication within the tumor and the release of tumorepitopes is not sufficient to cause antitumor response, however, andthus arming adenovirus with immunostimulatory molecules may augment theimmune responses against tumor antigens, thereby presenting a thirddanger signal.

Oncolytic adenoviruses that express granulocyte-macrophagecolony-stimulating factor (GM-CSF) induce anti-cancer immunity whileacting directly on cancer cells by oncolysis. GM-CSF is a potent inducerof systemic anti-tumor immunity associated with recruitment andmaturation of antigen presenting cells (APCs), mainly dendritic cells,as well as recruitment of cells of the innate immunity arm. However,systemically elevated cytokine levels represent a risk for toxicside-effects. Besides the direct risk of side effects mediated by highserums concentrations of GMCSF, an indirect risk results fromrecruitment of myeloid derived suppressor cells (MDSC). While theimmunosuppressive effect of MDSC is potentially harmful for cancerpatients in general, it could be particularly counterproductive in thecontext of cancer immunotherapy.

Further, the adenoviral E1A-protein is also toxic to cells. It istherefore important that therapeutic adenoviruses expressing GM-CSF canbe directed to cells in which the intended therapy is required andhealthy cells are left intact. Thus, there exists a great need to beable to control the replication of therapeutic oncolytic adenovirusescoding for GM-CSF. Other immunostimulatory molecules that could beexpressed from the viral backbone include CD40 ligand and monoclonalantibody against CTLA-4.

E3 promoter activation requires the transactivating function of E1Aprotein (Berk 1986). Thus, when E1A protein production is controlledunder the E2F-1 promoter, simultaneously an indirect control over E3gene products is elicited.

Another way which has been used to induce anti-cancer immunity isthrough CpG island which can be inserted into the E3 region downstreamfrom the GM-CSF transgene. Insertion of CpG dinucleotide islands intothe nucleotide backbone of the virus activates toll-like receptor 9(TLR9) expressed on B cells and dendritic cells (DC). Specifically,binding of CpG to TLR9 causes a conformational shift in the receptor,causing the recruitment of the adapter protein MyD88, activation ofsignaling pathways and subsequent activation of nuclear factor-κB(NF-κB) (Latz et al. 2007, Nat Immunol, 8, 772-779). On a cellular levelTLR9 activation initiates a cascade of innate and adaptive immuneresponses, such as activation of DCs and subsequent secretion ofchemokines, activation of NK-cells and expansion of T-cell populations,which may help initiate an immune response against infected tumor cells,and, via epitope spreading, against noninfected tumor cells as well.Previously CpG oligonucleotides have been studied as cancer vaccineadjuvants, but CpG islands have not been incorporated into oncolyticadenoviruses to enhance the immune reaction towards infected tumorcells. Since CpG islands represent patterns typical of microbes, it isunexpetected that they could be of utility in the context of treatmentof humans.

Modifications in the Fiber

Loss of CAR expression correlates with tumor progression, which implieslow expression levels of CAR in advanced disease (Okegawa et al. 2004).Cells expressing low levels of CAR are refractory to Ad5 infection, atleast in vitro. CAR dependency results in a scenario in which the targettissue of adenoviral gene therapy is poorly transduced, i.e. virusesenter target cells inefficiently, while non-target tissue with high CARexpression is efficiently transduced (Kim et al. 2002).

Fiber chimerism results in CAR binding ablation and alternate receptorrecognition, but is limited to the tropic behavior of the characterizedserotype adenoviruses. Increased transduction of ovarian cancer cellshas been achieved by replacing the Ad5 knob with the knob from Ad3(Krasnykh et al. 1996; Kanerva et al. 2002). Desmoglein 2 has recentlybeen identified as the primary receptor for serotype 3 adenoviruses(Wang et al. 2011, Nature Medicine, 17, 96-105). Desmoglein 2 is acalcium-binding transmembrane glycoprotein from the cadherin proteinfamily, and is a component of the cell-cell adhesion structure inepithelial cells. Desmoglein 2 is overexpressed in various epithelialmalignancies, including gastric, bladder and metastatic prostate canceras well as squamous cell carcinomas and melanoma (Biedermann et al.2005, J Pathol, 207, 199-206; Abbod et al. 2009, Expert Rev AnticancerTher, 9, 867-870; Trojan et al. 2005, Anticancer Res, 25, 183-191;Harada et al. 1996, Acta Derm Venereol, 76, 417-420; Schmitt et al.2007, J Invest dermatol, 127, 2191-2206).

Genetic modification of the capsid is a conceptually elegant approach toredirect adenoviral tropism. In short, the goal of genetic targeting isto create a single-component vector that can transduce cells vianon-native receptors. CAR independent gene delivery can be achieved byincorporating peptide ligands into the knob. This approach does notabrogate the native tropism, as the CAR binding ability is retained, butrather expands the vectors tropism. Ligands can be incorporated into twodistinct locales of the knob: the HI loop or the carboxy (C)-terminus.An RGD (arg-gly-asp)-ligand, targeting adenoviruses into integrins, or apolylysine (pK) motif, targeting them into heparan sulphateproteoglycans (HSPGs), have been incorporated into the C-terminus(Wickham et al. 1993, Cell 73:309-19; Borovjagin et al. 2005 Cancer GeneTher 12:475-86) and HI-loop (Krasnykh et al. 1998, J Virol 70:6839-46).In an aspect of the present invention the polylysine introduced in theC-terminus may comprise one or more than one lysine residues, e.g. from1 to 10 (Lys₁₋₁₀), 1 to 7 (Lys₁₋₇) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10lysines.

Further, Ad5 capsid in therapeutic adenoviruses has been modified toalter the biodistribution of systemically administered adenoviruses togain a more favorable tumor to liver ratio and, subsequently, lesstreatment related liver toxicity and inflammatory cytokine responses. Amutation in the putative HSPG binding motif (KKTK) of the Ad5 fibershaft has been shown to result in substantial reduction in livertransduction and inflammatory cytokine responses (Smith et al. 2003a,Hum Gene Ther 14, 777-87; Smith et al. 2003b, Human Gene Ther 14,1595-1604). However, all previous approaches exploring mutation of theHSPG region have been unable to retain or increase tumor targeting.Thus, reduction of liver uptake has been associated with reduction intumor uptake. It is quite surprising that the Ad3 knob in a KKTK mutatedAd5 fiber seems to be able to achieve this critical goal.

Despite attempts to develop effective adenoviral therapies with highsafety profile, there still remains a great demand and growing need forefficient and accurate gene transfer as well as increased specificityand sufficient tumor killing ability of gene therapies. The task ofdeveloping such therapies is particularly difficult because of safetyrequirements set for human therapies and the difficulties in avoidingclearance of the therapeutic virus due to host immune response. Of note,immunotherapies cannot be studied in vitro since an intact immune systemis required. Moreover, since human adenoviruses are quite speciesspecific, model systems do not capture the immunostimulatory effect ofoncolysis on anti-tumor immunity. This is compounded by the speciesspecificity of immunostimulatory molecules with regard to activity andsignaling. In other words, mouse GMCSF does not work in humans and viceversa, and mouse GMCSF works differently in mice than human GMCSF inhumans. Of all oncolytic adenovirus candidates tested today, only fewhave provided a level of therapeutic efficacy enabling successfulclinical use. This application describes strategies and provides methodsand means to both effectively recruit the host's immune system againstmalignant cells and simultaneously provide direct oncolytic activity inmalignant cells, while maintaining an excellent safety record.

Aspects of the invention are directed to novel methods and means forachieving efficient and accurate gene transfer as well as increasedspecificity and sufficient tumor killing ability in cancer gene therapy.The present application describes construction of recombinant viralvectors, methods relating to manufacturing said vectors, and use of saidvectors in tumor cells lines, animal models and cancer patients. Theinvention is also directed to host cells, compositions and kitscomprising said vectors.

In an aspect of the invention, the oncolytic adenoviruses according tothe invention can be used for improving, preventing and treating cancerin a subject.

In another aspect of the invention, the present invention relates tooncolytic adenoviruses derived from serotype 5 adenovirus. The inventiveadenoviruses have one, some or all of the following modifications intheir genome: the native E1A promoter controlling the expression E1Agene has been replaced by human E2F-1 promoter; a 24 base pair deletionhas been introduced to the E1A gene CR2 region, which results in adysfunctional E1A protein unable to bind cellular retinoblastoma proteinand subsequently release E2F transcription factors for the activation ofdownstream viral gene expression; 965 base pairs coding for the viralgenes gp19K and 6.7K have been deleted from the E3 region and atransgene GM-CSF has been introduced to replace them; the knob region ofthe fiber protein on the viral capsid has been replaced by a knob fromserotype 3 adenovirus resulting in a 5/3 chimeric fiber protein,enabling viral entry via desmoglein 2 protein instead of the nativereceptor for serotype 5 adenovirus, the coxsackie-adenovirus receptor(CAR). Alternatively, an RGD motif has been introduced into the HI loopof the native fiber knob or a polylysine (pK) motif has been introducedinto the C terminus of the native fiber knob. In combination with themodification of the knob a KKTK mutation has been introduced into theshaft of the fiber and a CpG island has been introduced in the E3region.

In another aspect of the invention, the present invention relates tooncolytic CGTG-602 adenovirus which comprises serotype 5 adenovirushaving the following modifications in the genome while the other regionsof the genome are intact: the native E1A promoter controlling theexpression E1A gene has been replaced by human E2F-1 promoter; a 24 basepair deletion has been introduced to the E1A gene CR2 region, whichresults in a dysfunctional E1A protein unable to bind cellularretinoblastoma protein and subsequently release E2F transcriptionfactors for the activation of downstream viral gene expression; 965 basepairs coding for the viral genes gp19K and 6.7K have been deleted fromthe E3 region and a transgene GM-C SF has been introduced to replacethem; the knob region of the fiber protein on the viral capsid has beenreplaced by a knob from serotype 3 adenovirus resulting in a 5/3chimeric fiber protein, enabling viral entry via desmoglein 2 proteininstead of the native receptor for serotype 5 adenovirus, thecoxsackie-adenovirus receptor (CAR).

In other aspects of the invention, the invention relates to followingserotype 5 based adenoviruses:

CGTG-601 is otherwise identical to CGTG-602, but it has an intactserotype 5 fiber.

CGTG-603 is otherwise identical to CGTG-601, but it has an RGD-4C motif(Arg-Gly-Asp and 4 cys) inserted in the HI-loop of the serotype 5 fiberknob. This modification enables viral entry to cells via cellularintegrins in addition to the native receptor CAR.

CGTG-604 is otherwise identical to CGTG-601, but it has seven lysinesintroduced to the C-terminus of the serotype 5 fiber knob. Thismodification enables viral entry to cells via heparin sulphateproteoglycans in addition to the native receptor CAR.

CGTG-605 is otherwise identical to CGTG-602, but it has CpG islandsinserted in the E3 downstream from the transgene GM-CSF.

CGTG-606 is otherwise identical to CGTG-602, but it has the KKTK motifof the serotype 5 fiber shaft mutated and replaced by a GAGA motif.

CGTG-607 is otherwise identical to CGTG-602, but it has CpG islandsinserted in E3 downstream from the transgene GM-CSF and the KKTK motifof the serotype 5 fiber shaft mutated and replaced by a GAGA motif.

In another aspect of the invention, the present invention provides amethod of treating cancer in a subject, wherein the method comprisesadministering the vector or pharmaceutical composition comprising thevector according to the invention to a subject, the method comprisingthe steps of

a) carrying a vehicle comprising an oncolytic adenoviral vector of theinvention to a cell, and

b) expressing GM-CSF of said vector in the cell.

In another aspect of the invention, the present invention provides amethod of increasing tumor specific immune response in a subject,wherein the method comprises:

a) carrying a vehicle comprising an oncolytic adenoviral vectoraccording to the invention to a target cell or tissue,

b) expressing recombinant GM-CSF of said vector in the target cell, and

c) Increasing amount of cytotoxic T cells and/or natural killer cells insaid target cell or tissue.

In another aspect of the invention, the present invention provides a useof the oncolytic adenoviral vector of the invention for producing GM-CSFin a cell.

In another aspect of the invention, the present invention provides anoncolytic adenoviral vector of the invention for producing GM-CSF in acell.

In another aspect of the invention, the present invention provides a useof the oncolytic adenoviral vector of the invention for increasing tumorspecific immune response in a subject.

In another aspect of the invention, the present invention provides anoncolytic adenoviral vector of the invention for increasing tumorspecific immune response in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are included to further demonstrate certainaspects and features of the present invention. The invention may bebetter understood by reference to one or more of these drawings incombination with the detailed description of specific aspects, includingexamples.

FIGS. 1A-1G. A schema of CGTG-602 (Ad5/3-E2F1.D24-GMCSF, SEQ ID NO:2)(FIG. 1A), CGTG-601 (Ad5-E2F.D24-GMCSF, SEQ ID NO:1) (FIG. 1B), CGTG-603(Ad5-RGD-E2F.D24-GMCSF, SEQ ID NO:3) (FIG. 1C), CGTG-604(Ad5-pK7-E2F.D24-GMCSF, SEQ ID NO:4) (FIG. 1D), CGTG-605(Ad5/3-E2F.D24-GMCSF-CpG, SEQ ID NO:5) (FIG. 1E) CGTG-606(Ad5/3-E2F1.D24-GMCSF-KKTK, SEQ ID NO:6) (FIG. 1F) and CGTG-607(Ad5/3-E2F1.D24-GMCSF-CpG-KKTK, SEQ ID NO:7) (FIG. 1G) genome. Theviruses have their E1A promoter replaced by a human E2F-1 promoter,which controls the transcription of the E1A gene. The E1A gene bears a24 base pair deletion in the constant region 2 to avoid theselfactivation of the promoter by E2F-1 released by E1A-Rb interaction,a critical fault in previous designs with an intact E1A gene. gp19k and6.7K in E3 have been replaced with the cDNA of human GM-CSF. ADP refersto the adenovirus death protein. CGTG-605 has a CpG dinucleotide islandinserted into the E3 region. CGTG-602 (SEQ ID NO: 2), CGTG-605 (SEQ IDNO:5) and CGTG-606 (SEQ ID NO:6) have a chimeric 5/3 fiber, where theserotype 5 (Ad5) knob has been replaced by the serotype 3 (Ad3) knob.Additionally, CGTG-606 (SEQ ID NO:6) has a KKTK motif replaced by GAGAon the fiber shaft. CGTG-607 (SEQ ID NO:7) has the features of CGTG-605and CGTG-606 combined. CGTG-603 (SEQ ID NO:3) has an RGD-4C (Arg-Gly-Aspand 4 X Cys) motif inserted in the HI-loop of the Ad5 knob. CGTG-604(SEQ ID NO:4) has a polylysine motif inserted into the C-terminus of theAd5 knob. CGTG-601 (SEQ ID NO:1) has a genetically intact Ad5 fiber.

FIG. 2 shows that adenovirus-expressed GMCSF retains its biologicalactivity in human lymphocytes. TF 1 cells, which require human GMCSF forstaying alive, were cultured in the presence of human recombinant GMCSF(E. coli-produced, purchased from Sigma) or supernatant from CGTG-602(a) or CGTG-603 (b) infected cells. The viability of TF1-cells treatedwith recombinant hGMCSF was set as 100%.

FIG. 3 shows that replacing the native E1A promoter with E2F-1 promoterdoes not impair virus replication and cell killing effect in vitro. FIG.3 represents results of MTS assay showing cell killing efficiency of thenovel virus CGTG-602 compared to CGTG-102—a control virus bearing thegenetically intact E1A promoter—a wild type serotype 5 virus and anon-replicating control virus Ad5/3 lucl in A549 lung cancer cells,SKOV3ip.1 ovarian cancer cells and PC3-MM2 prostate cancer cells.

FIG. 4 shows that the E2F-1 promoter improves the in vitro selectivityof CGTG-602 replication. Human primary hepatocytes were infected withCGTG-602 or control viruses CGTG-102, Ad5 wt, Ad5/3-D24-Cox2L ormock-infected. Cells and supernatant were collected 24, 48 or 72 hourslater and analyzed for infective virus particles by standard plaqueassay on 293 cells.

FIG. 5 shows in vivo selectivity of CGTG-602 in Syrian hamsters.CGTG-602 was injected intratumorally in HAPT-1 pancreatic cancer tumorsor straight into livers of animals without tumors and the tumors orlivers were collected 0.5, 24, 48, 72 or 96 hours later and the amountof viral DNA was analyzed by qPCR. Results are depicted as viral E4 copynumbers relative to hamster genomic dna (ng).

FIG. 6 shows in vivo efficiency of CGTG-602 in Syrian Hamsters(semipermissive for human adenovirus replication) bearing pancreaticcancer tumors. CGTG-602 significantly (P<0.01) slowed down tumorprogression, and was more potent than the control virus CGTG-102. 3×10⁸VP of virus was administered intratumorally on days 0, 3 and 6. Thesmallest tumors were seen in the group which received concomitantlow-dose cyclophosphamide (2 mg/hamster).

FIG. 7A shows in vivo efficiency of CGTG-603 in Syrian Hamsters bearingpancreatic cancer tumors. 1×10⁹ VP of virus was administered on days 0,2 and 4. Ad5-D24-RGD (lacks E2F-1 promoter and GM-CSF gene),Ad5-RGD-D24-GMCSF (lacks E2F-1 promoter) and Ad5 wt were used as controlviruses. Mock treated animals received growth medium only. All viruseseradicate the tumors within 16 days following the treatments.

FIGS. 7B-7C show a rechallenge experiment with the same viruses and aGM-CSF producing ICOVIR-15 virus (an adenovirus with a modified E2F-1promoter and an RGD-modified capsid) as an additional control virus.Hamster HAPT-1 tumors were treated intratumorally with 1×10⁹ VP of eachvirus as described above and tumor growth was followed for 20 days (FIG.7B). Tumors were surgically removed, hamsters were re-challenged withHAPT-1 tumors and re-treated (FIG. 7C). All hamsters treated with virusshowed delay in tumor growth, but only those hamsters that had beenpreviously treated with CGTG-603 or one of the other viruses containingGM-CSF as a transgene showed a complete protection against tumorre-challenge.

FIGS. 8A-8B show CGTG-602 induced CD8+ T-cell infiltration in (FIG. 8A)human tumor tissue but not in (FIG. 8B) normal peritoneal liningobtained from patient C332 1 month after treatment with CGTG-602. Cellsstained positive for CD8 appear as brown.

FIGS. 9A-9B show that CGTG-602 elicited a T cell-response against bothtumor epitopes and adenovirus (present in tumor cells). PBMCs harvestedfrom patients treated with CGTG-602 were analyzed by IFN-gamma ELISPOTupon stimulation with a mix of peptide from Adenovirus 5 and mixes ofpeptides from tumor antigens CEA and NY-ESO-1 (pooll), c-myc and SSX2(pool2) and surviving alone. FIG. 9A represents the frequency ofIFN-gamma producing peptide specific PBMCs, FIG. 9B the proportion ofthe peptide specific IFN-gamma producing PBMC from all activated PBMC(per million cells.

FIG. 10 shows the results obtained when antibodies against tumorassociated antigens (TAAs) NY-ESO-1, CA-15-3, CEA and survivin wereanalyzed from patient serum (a) and ascites fluid and cells of patient0314 (b) before and after viral treatment, and the data is presented asproportional change (%) of antibody levels from pre-treatment value. (a)Treatment often resulted in decrease of elevated levels of antibodiesagainst TAAs in patients that showed a concomitant decrease in markerlevels (patients O314, R356, R319, O340). (b) Malignant ascites(resulting from peritoneal tumor masses) was removed from the peritonealcavity of patient O314 before and 19 and 40 days after virusadministration. Results were compared to control serum levels and datafor those TAAs that were elevated at any time point are shown.

FIG. 11 shows the patient serum marker levels for CEA, Ca12-5, Ca15-3and/or Ca 19-9 after treatment.

FIG. 12 shows a survival plot for CGTG-602 treated patients. beforetreatment, FIG. 13B after treatment) and a 49.1% reduction in themetabolic activity of an injected liver lesion (FIG. 13C beforetreatment, FIG. 13D after treatment) of patient R319.

FIGS. 14A-14F show positron emission tomography-computed tomography(PET-CT) fusion images from patients R319 and 5354. (FIG. 14A) baseline,(FIG. 14B) after 3 months of treatment, (FIG. 14C) after 6 months, (FIG.14D) after 9 months. Tumor size started diminishing at 6 months. (FIGS.14E-14F) a different plane of PET analysis from the same patient atbaseline and after 3 months, arrows indicate PET active regions of thetumor.

FIG. 15 shows that the mutation of the KKTK motif significantly reducesthe viral transduction to liver (a), but contrary to previous reportsdoes not significantly alter the transduction efficacy to tumors (b).Briefly, a non-replicating virus with a 5/3 chimeric capsid was used forintravenous injections in tumor bearing mice and the viral load intissue was quantitated by qPCR.

FIG. 16 shows that the mutation of the KKTK motif of a 5/3 chimericvirus results in transduction of cancer cell lines Hey (a), PC-3 (b),SKOV3.ipl and M4A4-LMN3 (c) to similar degree as a virus with anunmodified capsid and in some case to similar degree as Ad5/3 Lucl.

FIG. 17 shows that the CpG island induces NFkB activation via TLR-9 in293-hTLR9 cells. A represents the actual luciferase signal from theimaging and B shows the results in a bar graph.

FIGS. 18A-18B show antibody levels against serotype 5 hexon analyzedfrom serum of all patients (FIG. 18A) and ascites cells and fluid ofpatient 0314 (FIG. 18B) before and after viral treatment. (FIG. 18A) Inall patients, serum levels were above weakly positive value of 35 U/mlvalue before treatment but were dramatically elevated after treatment.(FIG. 18B) Ascites cells from patient 0314 showed a value of 43 U/100 mgbefore and 471 U/100 mg after treatment, suggesting presence of CGTG-602virus in the malignant cells. Ascites fluid values were elevated from 58U/ml before treatment to 758 U/ml after treatment.

SEQUENCE LISTINGS

SEQ ID NO: 1 is the nucleotide sequence encoding the virus CGTG-601.

SEQ ID NO: 2 is the nucleotide sequence encoding the virus CGTG-602.

SEQ ID NO: 3 is the nucleotide sequence encoding the virus CGTG-603.

SEQ ID NO: 4 is the nucleotide sequence encoding the virus CGTG-604.

SEQ ID NO: 5 is the nucleotide sequence encoding the virus CGTG-605.

SEQ ID NO: 6 is the nucleotide sequence encoding the virus CGTG-606.

SEQ ID NO: 7 is the nucleotide sequence encoding the virus CGTG-607.

SEQ ID NO: 8 is the nucleotide sequence encoding the plasmidpE2F.E1.D24.

SEQ ID NO: 9 is the nucleotide sequence encoding the plasmidpAd5/3-E2F-D24-GMCSF.

SEQ ID NO: 10 is the nucleotide sequence of the primer E4-forward.

SEQ ID NO: 11 is the nucleotide sequence of the primer E4-reverse.

SEQ ID NO: 12 is the nucleotide sequence of the probe E4.

SEQ ID NO: 13 is the nucleotide sequence of the primer GAPDH-forward.

SEQ ID NO: 14 is the nucleotide sequence of the primer GAPDH-reverse.

SEQ ID NO: 15 is the nucleotide sequence of the probe GAPDH.

SEQ ID NO: 16 is the nucleotide sequence of the E1-forward primer.

SEQ ID NO: 17 is the nucleotide sequence of the E1-reverse primer.

SEQ ID NO: 18 is the nucleotide sequence of the probe “onco”.

SEQ ID NO: 19 is the nucleotide sequence of the probe “wt”.

SEQ ID NO: 20 is the nucleotide sequence of the GM-CSF-forward.

SEQ ID NO: 21 is the nucleotide sequence of GM-CSF-reverse primer.

SEQ ID NO: 22 is the nucleotide sequence of human beta-actin-forwardprimer.

SEQ ID NO: 23 is the nucleotide sequence of human beta-actin-reverseprimer.

SEQ ID NO: 24 is the nucleotide sequence of human beta-actin probehaving 6FAM marker in the 5′ end and TAMRA marker in the 3′ end.

SEQ ID NO: 25 is the nucleotide sequence of mouse beta-actin-forwardprimer.

SEQ ID NO: 26 is the nucleotide sequence of mouse beta-actin-reverseprimer.

SEQ ID NO: 27 is the nucleotide sequence of mouse beta-actin probehaving 6FAM marker in the 5′ end and TAMRA marker in the 3′ end.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used inthis application have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Standardone-letter notations for nucleic acids and three-letter and one-letternotations for amino acids are used interchangeably herein.

As used herein, the expression “adenovirus serotype 5 (Ad5) nucleic acidbackbone” or “virus genome” refers to the genome or partial genome ofAd5, which comprises one or several regions selected from the groupconsisting of partial E1, pIX, pIVa2, E2, VA1, VA2, L1, L2, L3, L4,partial E3, L5 and E4 of Ad5 origin. One preferred vector of theinvention comprises nucleic acid backbone of Ad5. In another preferredvector, the adenoviral nucleic acid backbone is mostly derived from Ad5and combined with a portion (e.g. a part of the capsid structure) ofAd3.

As used herein, expression “partial” as used in the context of regionsof viral DNA refers to a region, which lacks any part compared to acorresponding wild type virus region. “Partial E1” refers to E1 regionwith D24 deletion and “partial E3” refers to E3 region lackinggp19k/6.7K.

As used herein, the terms “VA1” and “VA2” refer to virus associated RNAs1 and 2, which are transcribed by the adenovirus but are not translated.VA1 and VA2 have a role in combating cellular defence mechanisms.

As used herein, the expression “a viral packaging signal” refers to apart of virus DNA, which consists of a series of AT-rich sequences andgoverns the encapsidation process.

As used herein the term “capsid” refers to the protein shell of thevirus, which includes hexon, fiber and penton base proteins.

As used herein, the expression “Ad5/3 chimerism” of the capsid refers toa chimerism, wherein the knob part of the fiber is from Ad serotype 3,and the rest of the fiber is from Ad serotype 5.

As used herein, the expression “RGD region” refers to thearginine-glycine-aspartic acid (RGD) motif, which is exposed on thepenton base and interacts with cellular av integrins supportingadenovirus internalization.

As used herein, a “KKTK mutation” refers to a nucleotide sequenceaaaaaaaccaag (base pairs 30892-30903, translating into KKTK) replaced bynucleotide sequence ggagccggagcc (translating into GAGA).

As used herein, the expression “expression cassette” refers to a DNAvector or a part thereof comprising nucleotide sequences, which encodecDNAs or genes, and nucleotide sequences, which control and/or regulatethe expression of said cDNAs or genes. Similar or different expressioncassettes may be inserted to one vector or to several different vectors.Ad5 vectors of the present invention may comprise either several or oneexpression cassettes.

The term “mutation” as used herein refers to a deletion, an insertion ofnucleic acid, an inversion, or a substitution as commonly understood inthe art.

The term “gene” as used herein refers to a segment of nucleic acid thatencodes an individual protein or RNA (also referred to as a “codingsequence” or “coding region”), optionally together with the associatedregulatory regions such as promoters, operators, terminators and thelike, that may be located upstream or downstream of the coding sequence.

The terms “mutant virus”, “modified virus” and “modified virus vector”as used herein refer to a virus comprising one or more mutations in itsgenome, including but not limited to deletions, insertions of nucleicacids, inversions, substitutions or combinations thereof.

The term “naturally-occurring”, “native” and similar expressions as usedherein with reference to a virus indicates that the virus is in the formin which it can be found in nature, i.e. it can be isolated from asource in nature in this form and it has not been intentionallymodified.

The term “wild-type virus” as used herein refers to the most frequentgenotype of a virus found in nature and against which mutants aredefined.

The term “anti-viral response” as used herein refers to a cell'sresponse to viral infection and includes, for example, production ofinterferons, cytokine release, production of chemokines, production oflymphokines or a combination thereof

The expressions “normal host cell” and “normal tissue” as used hereinrefer to a non-cancerous, non-infected cell or tissue with an intactanti-viral response.

The term “oncolytic agent” as used herein refers to an agent capable ofinhibiting the growth of and/or killing tumour cells.

The term “subject” as used herein refers to any living organism,including humans and animals, human and animal tissue, and human andanimal cells.

An object of the present invention was to develop novel therapeuticallyeffective oncolytic adenoviruses with improved safety properties forcancer therapy and to solve problems encountered in conventional cancertherapy and in cancer virotherapy.

The inventors have surprisingly found an oncolytic adenoviral vectorwhich is both therapeutically effective and safe in use. The recombinantadenovirus according to the invention comprises one or more of thefollowing elements: an adenovirus serotype 5 (Ad5) nucleic acidbackbone; a nucleic acid sequence encoding a tumor specific human E2F-1promoter replacing the E1A promoter for the control of the of E1A genetranscription; a 24 by deletion (D24) in the Rb binding constant region2 of adenoviral E1; a nucleic acid sequence encoding agranulocyte-macrophage colony-stimulating factor (GM-CSF) in the placeof the deleted gp19k/6.7K in the adenoviral E3 region; a nucleic acidsequence replacing the serotype 5 adenoviral fiber knob region with thatof a serotype 3 adenovirus knob region; with or without a nucleic acidsequence comprising CpG island in the E3 region after GMCSF-gene; withor without a mutated KKTK-region in the fiber shaft region. Theunexpected efficacy of the inventive oncolytic adenovirus provides asignificant improvement in the therapeutic efficacy as demonstrated inin vivo studies in comparison to previous oncolytic adenoviruses. Thesafety record of the inventive oncolytic adenovirus is also excellent.Efficacy and safety of the agents was found to be unexpectedly goodespecially in human patients.

In some aspects, the present invention provides cells comprising theadenoviral vector of the invention.

In some aspects, the present invention provides a pharmaceuticalcomposition comprising adenoviral vectors of the invention. Apharmaceutical composition of the invention comprises at least one typeof the vectors of the invention. Furthermore, the composition maycomprise at least two, three, four or more different vectors of theinvention. In addition to the vector of the invention, a pharmaceuticalcomposition of the invention may comprise any other vectors, such asother adenoviral vectors, other therapeutically effective agents, anyother agents such as pharmaceutically acceptable carriers, buffers,excipients, adjuvants, antiseptics, filling, stabilising or thickeningagents, and/or any components normally found in corresponding products.

The pharmaceutical composition may be in any form, such as solid,semisolid or liquid form suitable for administration. A formulation canbe selected from a group consisting of, but not limited to, solutions,emulsions, suspensions, tablets, pellets and capsules.

In an aspect of the invention, the oncolytic adenoviral vector orpharmaceutical composition acts as an in situ cancer vaccine. As usedherein “in situ cancer vaccine” refers to a cancer vaccine, which bothkills tumor cells and also increases the immune response against tumorcells. Virus replication is a strong danger signal to the immune system(=needed for a TH1 type response), and thus acts as a powerfulcostimulatory phenomenon to GM-CSF mediated maturation and activation ofAPCs, and recruitment of NK cells. Tumor cell lysis also helps topresent tumor fragments and epitopes to APCs and furthermore,costimulation is produced by inflammation. Thus, an epitope independent(i.e. not HLA restricted) response is produced in the context of eachtumor and therefore takes place in situ. Tumor specific immune responseis activated in the target cell as well as the surrounding cells, e.g.in the target tissue.

The effective dose of vectors depends on at least the subject in need ofthe treatment, tumor type, location of the tumor and stage of the tumor.The dose may vary for example from about 10e8 viral particles (VP) toabout 10e14 VP, preferably from about 5×10e9 VP to about 10e13 VP andmore preferably from about 8×10e9 VP to about 10e12 VP. In one specificaspect of the invention the dose is in the range of about5×10e10-5×10e11 VP.

The pharmaceutical compositions may be produced by any conventionalprocesses known in the art, for example by utilizing any one of thefollowing: batch, fed-batch and perfusion culture modes,column-chromatography purification, CsCl gradient purification andperfusion modes with low-shear cell retention devices.

The vector or pharmaceutical composition of the invention may beadministered to any eukaryotic subject selected from the groupconsisting of plants, animals and human beings. In a preferred aspect ofthe invention, the subject is a human or an animal. An animal may beselected from a group consisting of pets, domestic animals andproduction animals. In an aspect of the invention the subject is human.

Any conventional method may be used for administration of the vector orcomposition to a subject. The route of administration depends on theformulation or form of the composition, the disease, location of tumors,the patient, comorbidities and other factors. In a preferred aspect ofthe invention, the administration is conducted through an intratumoral,intramuscular, intra-arterial, intravenous, intrapleural,intravesicular, intracavitary or peritoneal injection, or an oraladministration. Therapeutic compositions are formulated relative to theparticular administration route.

In an aspect, in the present invention oncolytic adenoviral vectors areadministered in a single administration to achieve therapeutic effects.However, in a preferred aspect of the invention, oncolytic adenoviralvectors or pharmaceutical compositions are administered several timesduring the treatment period. Oncolytic adenoviral vectors orpharmaceutical compositions may be administered for example from 1 to 10times in the first 2 weeks, 4 weeks, monthly or during the treatmentperiod. In an aspect of the invention, administration is done three toseven times in the first 2 weeks, then at 4 weeks and then monthly. Inan aspect of the invention administration is done four times in thefirst 2 weeks, then at 4 weeks and then monthly. The length of thetreatment period may vary, and for example may last from two to 12months or more.

In order to avoid neutralizing antibodies in a subject, the vectors ofthe invention may vary between treatments. In a preferred aspect of theinvention, the oncolytic adenoviral vector having a different fiber knobof the capsid compared to the vector of the earlier treatment isadministered to a subject. As used herein “fiber knob of the capsid”refers to the knob part of the fiber protein (FIGS. 1A-1G).

The therapy of the invention is effective alone, but combination ofadenoviral gene therapy with other therapies, such as traditionaltherapy, may be more effective than either one alone. For example, eachagent of the combination therapy may work independently in the tumortissue, the adenoviral vectors may sensitize cells to chemotherapy orradiotherapy and/or chemotherapeutic agents may enhance the level ofvirus replication or effect the receptor status of the target cells. Theagents of combination therapy may be administered simultaneously orsequentially.

In a preferred aspect of the invention, the method or use furthercomprises administration of concurrent radiotherapy to a subject. Inanother preferred aspect of the invention, the method or use furthercomprises administration of concurrent chemotherapy to a subject. Asused herein “concurrent” refers to a therapy, which has beenadministered before, after or simultaneously with the gene therapy ofthe invention. The period for a concurrent therapy may vary from minutesto several weeks. Preferably the concurrent therapy lasts for somehours.

Agents suitable for combination therapy or which can be used as virussensitizers include but are not limited to All-trans retinoic acid,Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine,Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone,erlotinib, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea,Idarubicin, Imatinib, Mechlorethamine, Mercaptopurine, Methotrexate,Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Temozolomide,Teniposide, Tioguanine, Valrubicin, Vinblastine, Vincristine, Vindesineand Vinorelbine.

In a preferred aspect of the invention, the method or use furthercomprises administration of verapamil or another calcium channel blockerto a subject. “Calcium channel blocker” refers to a class of drugs andnatural substances which disrupt the conduction of calcium channels, andit may be selected from a group consisting of verapamil,dihydropyridines, gallopamil, diltiazem, mibefradil, bepridil,fluspirilene and fendiline.

In a preferred aspect of the invention, the method or use furthercomprises administration of autophagy inducing agents to a subject.Autophagy refers to a catabolic process involving the degradation of acell's own components through the lysosomal machinery. “Autophagyinducing agents” refer to agents capable of inducing autophagy and maybe selected from a group consisting of, but not limited to, mTORinhibitors, PI3K inhibitors, lithium, tamoxifen, chloroquine,bafilomycin, temsirolimus, sirolimus and temozolomide. In a specificaspect of the invention, the method further comprises administration oftemozolomide to a subject. Temozolomide may be either oral orintravenous temozolomide.

In one aspect of the invention, the method or use further comprisesadministration of chemotherapy or anti-CD20 therapy or other approachesfor blocking of neutralizing antibodies. “Anti-CD20 therapy” refers toagents capable of killing CD20 positive cells, and may be selected froma group consisting of rituximab and other anti-CD20 monoclonalantibodies. “Approaches for blocking of neutralizing antibodies” refersto agents capable of inhibiting the generation of anti-viral antibodiesthat normally result from infection and may be selected from a groupconsisting of different chemotherapeutics, immunomodulatory substances,corticoids and other drugs. These substances may be selected from agroup consisting of, but not limited to, cyclophosphamide, cyclosporin,azathioprine, methylprenisolone, etoposide, CD4OL, CTLA4Ig4, FK506(tacrolismus), IL-12, IFN-gamma, interleukin 10, anti-CD8, anti-CD4antibodies, myeloablation and oral adenoviral proteins.

The oncolytic adenoviral vector of the invention induces virion mediatedoncolysis of tumor cells and activates human immune response againsttumor cells. In a preferred aspect of the invention, the method or usefurther comprises administration of substances capable of downregulatingregulatory T-cells in a subject. “Substances capable of downregulatingregulatory T-cells” refers to agents that reduce the amount of cellsidentified as T-suppressor or Regulatory T-cells. These cells have beenidentified as consisting one or many of the following immunophenotypicmarkers: CD4+, CD25+, FoxP3+, CD127− and GITR+. Such agents reducingT-suppressor or Regulatory T-cells may be selected from a groupconsisting of anti-CD25 antibodies or chemotherapeutics.

In a preferred aspect of the invention, the method or use furthercomprises administration of cyclophosphamide to a subject.Cyclophosphamide is a common chemotherapeutic agent, which has also beenused in some autoimmune disorders. In the present invention,cyclophosphamide can be used as a virus sensitizer to enhance viralreplication and the effects of GM-CSF induced stimulation of NK andcytotoxic T-cells for enhanced immune response against the tumor. It canbe used as intravenous bolus doses or low-dose oral metronomicadministration. Other suitable virus sensitizers that can be used inaspects of present invention include temozolomide and erlotinib.

Any method or use of the invention may be either in vivo, ex vivo or invitro method or use.

The present invention also relates to a method of treating cancer in asubject, wherein the method comprises administering the vector orpharmaceutical composition of the invention to a subject; carrying avehicle comprising an oncolytic adenoviral vector of the invention intoa cell; and expressing GM-CSF of said vector in the cell.

Furthermore, the present invention relates to a method of increasingtumor specific immune response in a subject, wherein the methodcomprises carrying a vehicle comprising an oncolytic adenoviral vectorof the invention to a target cell or tissue; expressingimmunostimulatory GM-CSF of said vector in the cell; and increasingamount of cytotoxic T cells and/or natural killer cells in said targetcell or tissue.

Furthermore, the present invention provides use of the oncolyticadenoviral vector of the invention for producing GM-CSF in a cell.

Furthermore, the present invention provides an oncolytic adenoviralvector for producing GM-CSF in a cell.

Furthermore, the present invention provides use of the oncolyticadenoviral vector for increasing tumor specific immune response in asubject.

Furthermore, the present invention provides oncolytic adenoviral vectorfor increasing tumor specific immune response in a subject.

Furthermore, the present invention provides tool for treatment ofcancers, which are refractory to current approaches. Also, restrictionsregarding tumor types suitable for treatment remain few compared to manyother treatments. In fact all solid tumors may be treated with thepresent invention. Larger tumors by mass and more complex tumors can becured by the present invention. The treatment can be givenintratumorally, intracavitary, intravenously and in a combination ofthese. The approach can give systemic efficacy despite local injection.The approach can also eradicate cells proposed as tumor initiating(“cancer stem cells”).

In an aspect of the invention the oncolytic adenoviral vector comprisesa human E2F-1 promoter replacing the viral E1A promoter upstream of theE1A region, lacks 24 base pairs from CR2 in E1A gene, and gp19k and 6.7Kin E3 region, and comprises a human GM-CSF in place of the deletedsequence in E3.

In another aspect of the invention, the adenoviral vector of theinvention comprises a capsid modification in the fiber of the virus, aCpG island in the E3 region and/or a KKTK mutation in the fiber gene inthe shaft region.

In another aspect of the invention, in addition to partial regions E1and E3, the oncolytic adenoviral vector of the invention may furthercomprise one or more regions or elements selected from the groupconsisting of viral early genes, viral intermediate genes and viral lategenes, preferably E2, E4, and late regions.

In another aspect of the invention, the oncolytic adenoviral vectorcomprises the following regions or elements: a left ITR, partial E1,pIX, pIVa2, E2, VA1, VA2, L1, L2, L3, L4, partial E3, L5, E4, and aright ITR. In another aspect of the invention the regions or elementsmay be in any order in the vector, but in a preferred aspect the regionsare in a sequential order in the 5′ to 3′ direction. Open reading frames(ORFs) may be in the same DNA strand or in different DNA strands. In apreferred aspect of the invention, the E1 region comprises a viralpackaging signal.

In an aspect of the invention, a gene encoding an immunostimulatoryprotein, preferably human GM-CSF, is incorporated in the virus vectorfor immunostimulatory effect. As is obvious to persons skilled in theart, other proteins exerting similar immunostimulatory effect and beingpharmaceutically acceptable, such as genes encoding human CD40 ligandand human anti-CTLA-4 antibody may also be used in the vector instead ofGM-CSF.

In an aspect the present invention provides recombinant serotype 5 (Ad5)adenovirus being capable of replicating and having lytic activity intarget cells wherein the virus comprises in the genome thereof a nucleicacid sequence encoding a target cell specific promoter replacing thenatural E1A adenoviral promoter; at least one modification in the Rbbinding constant region 2 of adenoviral E1 disrupting the ability tobind Rb and preventing virus replication outside target cells; at leastone modification in the viral E3 genes disrupting the ability to controlhost immune response; and a nucleic acid sequence encoding animmunostimulatory protein operably linked to the promoter of adenoviralE3.

In an embodiment the present invention provides a recombinant serotype 5(Ad5) adenovirus being capable of replicating and having lytic activityin target cells characterized in that the virus comprises in the genomethereof a nucleic acid sequence encoding E2F-1 promoter replacing thenatural E1A adenoviral promoter; at least a 24 by deletion (D24) in theRb binding constant region 2 of adenoviral E1 disrupting the ability tobind Rb and preventing virus replication outside target cells; at leasta deletion in gp19k/6.7K in any of the viral E3 genes disrupting theability to control host immune response and an insertion of animmunostimulatory transgene in the deleted region operably linked to thepromoter of adenoviral E3; and a nucleic acid element which activatesTLR9.

In an aspect the present invention provides recombinant Ad5 adenovirusabove wherein the nucleic acid sequence encoding a target cell specificpromoter replacing the natural E1A adenoviral comprises E2F-1 promoter.

In an aspect the present invention provides recombinant Ad5 adenovirusabove, wherein the at least one modification in the Rb binding constantregion 2 of adenoviral E1 disrupting the ability to bind Rb comprises a24 by deletion (D24) in the Rb binding constant region 2 of theadenoviral E1.

In an aspect the present invention provides recombinant Ad5 adenovirusabove, wherein the at least one modification in any of the viral E3genes disrupting the ability to control host immune response comprises adeletion in gp19k/6.7K of the adenoviral E3 region comprising the viralE3 genes and an operably linked insertion of an immunostimulatorytransgene, preferably a GM-C SF, in the deleted region.

In an aspect the present invention provides recombinant Ad5 adenovirusabove, wherein the adenovirus genome comprises a capsid modification.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the capsid modification is a fiber knob regionsubstitution wherein a region encoding Ad5 adenoviral fiber knob isreplaced by the corresponding region from another adenovirus serotype,preferably from Ad3.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the adenovirus genome comprises a nucleic acidelement which activates TLR9.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the nucleic acid element which activates TLR9comprises CpG island inserted in the E3 region downstream of thetransgene encoding the immunostimulatory protein.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein when the fiber knob region comprises Ad5adenoviral fiber knob the adenovirus genome comprises an RGD motif(Arg-Gly-Asp) inserted in the HI loop of the adenoviral fiber knob.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein when the fiber knob region comprises Ad5adenoviral fiber knob the adenovirus genome comprises a polylysine motif(Lys₁₋₇) introduced in the C terminus of the fiber knob.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein when the fiber knob region comprises Ad3adenoviral fiber knob, the nucleic acid modification in the fiber shaftregion comprises a mutation in a KKTK motif, preferably a GAGA motifsubstituted for the KKTK motif.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the virus genome further comprises at leastone element selected from the group consisting of viral immediate earlygenes, intermediate genes, and late genes.

In an aspect the present invention provides the recombinant Ad5adenovirus above for use in therapy.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the therapy is for treating and/or preventingany condition susceptible of being improved or prevented by saidrecombinant Ad5 adenovirus.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the therapy is for cancer.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the cancer is selected from a group consistingof nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renalcancer, cancer of connective tissues, melanoma, lung cancer, bowelcancer, colon cancer, rectal cancer, colorectal cancer, brain cancer,throat cancer, oral cancer, liver cancer, bone cancer, pancreaticcancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma,T-cell leukemia/lymphoma, neuroma, von HippelLindau disease,Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile ductcancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma,meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing'ssarcoma, cancer of unknown primary site, carcinoid, carcinoid ofgastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease,cervical cancer, esophagus cancer, gall bladder cancer, head cancer, eyecancer, neck cancer, kidney cancer, Wilms' tumor, Kaposi's sarcoma,prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin'slymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer,endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroidcancer, penis cancer, pituitary cancer, soft tissue sarcoma,retinoblastoma, small intestine cancer, stomach cancer, thymus cancer,thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer,endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma,mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gumcancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve 5cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynxcancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsilcancer.

In an aspect the present invention provides the recombinant Ad5adenovirus above, wherein the virus comprises the nucleic acid sequenceaccording to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9.

In an aspect the present invention provides a method of producingrecombinant Ad5 adenovirus particles, wherein the method comprisesproviding recombinant Ad5 adenovirus above inside a host cell permissivefor adenovirus replication, culturing the host cells under conditionsallowing said recombinant Ad5 adenovirus to propagate to formrecombinant Ad5 adenovirus particles, and recovering said infectiousrecombinant Ad5 adenovirus particles.

In an aspect the present invention provides a pharmaceutical compositionwhich comprises the recombinant Ad5 adenovirus above and apharmaceutically acceptable carrier.

In an aspect the present invention provides a virus particle whichcomprises the recombinant Ad5 adenovirus above.

In an aspect the present invention provides a host cell which comprisesthe recombinant Ad5 adenovirus above or the virus particle above.

In an aspect the present invention provides the recombinant Ad5adenovirus above, the pharmaceutical composition above, the virusparticle above, or the host cell above for inducing immunity directedagainst cancer, treating tumors and/or preventing tumors.

In an aspect the present invention provides a method of using therecombinant Ad5 adenovirus above, the pharmaceutical composition above,the virus particle above, or the host cell above for inducing immunitydirected against cancers, treating tumors and/or preventing tumors.

In an aspect the present invention provides a method of cancer therapywherein the method comprises administering the recombinant Ad5adenovirus above, the pharmaceutical composition above, the virusparticle above, or the host cell above to a subject.

In an aspect the present invention provides a method for manufacturing amedicament for therapy intended for any condition susceptible of beingimproved or prevented by administering said medicament, wherein therecombinant Ad5 adenovirus above, the virus particle above, or the hostcell above is used.

In an aspect the present invention provides the recombinant Ad5 serotypeadenovirus above or the method of cancer therapy above, wherein therecombinant Ad5 adenovirus is administered several times.

In an aspect the present invention provides the recombinant Ad5 serotypeadenovirus above or the method of cancer therapy above, wherein therecombinant Ad5 adenovirus is administered several times and theadenovirus used in subsequent administrations is different from therecombinant Ad5 adenovirus used in the first administration.

In an aspect the present invention provides the recombinant Ad5 serotypeadenovirus according to any one of claims 13-17 or the method of cancertherapy above, wherein the therapy comprises radiotherapy, surgery, oradministering one ore more agent selected from the group consisting of avirus sensitizer, chemotherapeutic agent, verapamil, calcium channelblocker, anti-CD20 therapy, and autophagy inducing agent.

Besides enabling the transport of the vector to the site of interest theadenovirus vector of the invention also assures the expression andpersistence of the transgene. Furthermore, immune response against thevector as well as the transgene is minimized.

The present invention solves problems related to therapeutic resistanceto conventional treatments. Furthermore, the present invention providestools and methods for selective treatments, with less toxicity ordamages in healthy tissues. Advantages of the present invention includealso different and reduced side effects in comparison to othertherapeutics. Importantly, the approach is synergistic with many otherforms of therapy including chemotherapy and radiation therapy, and istherefore suitable for use in combination regimens.

Induction of an immune reaction towards cells that allow replication ofunarmed viruses is normally not strong enough to lead to development oftherapeutic tumor immunity. In order to overcome this weakness, thepresent invention provides armed viruses with a potent inducer ofanti-tumor immunity. The present invention achieves cancer therapy,wherein tumor cells are destroyed by virion caused oncolysis. Inaddition, various different mechanisms activating human immune response,including activation of natural killer cells (NK) and dendritic cells(DC) are recruited for therapeutic use in the present invention.

Compared to adenoviral tools of the prior art, the present inventionprovides a more simple, more effective, inexpensive, non-toxic and safertool for cancer therapy. Furthermore, the present invention makes itunnecessary to use any helper viruses that are required in prior viraltherapies.

The novel products of the invention enable further improvements incancer therapy.

EXAMPLES

The following examples are given solely for the purpose of illustratingvarious aspects of the invention and they are not meant to limit thepresent invention. One skilled in the art will appreciate readily thatthe present invention is well adapted to carry out the objects andobtain the aims and advantages mentioned above, as well as thoseobjects, aims and advantages inherent herein. Changes therein and otheruses which are encompassed within the spirit of the invention as definedby the scope of the claims will occur to those skilled in the art.

Example 1 Cloning of CGTG-602 (SEQ ID NO:2)

CGTG-602 was constructed as follows. A pAdEasy-1-derived plasmidcontaining a chimeric 5/3 fiber, pAdEasy5/3, was created by homologousrecombination in E. coli of Ad5/3 lucl viral genome and BstXI-digested8.9 kb fragment of pAdEasy-1. Next, a shuttle vector containing a 24-bpdeletion in E1A (pShuttleD24) was linearized with PmeI and recombinedwith pAdEasy5/3 resulting in pAd5/3-D24. In order to insert human GMCSFgene into E3 region, an E3-cloning vector pTHSN was created by insertingSpel to NdeI fragment from Ad5 genome into the multi-cloning site ofpGEM5Zf+ (Promega, Madison, Wis.). pTHSN was further digested withSunI/MunI creating a 965-bp deletion in E3 region (6.7K and gp19Kdeleted) (described in Kanerva et al. 2005, Gene Ther 12:87-94). The 432by cDNA encoding human GMCSF (Invitrogen, Carlsbad Calif.) was amplifiedwith primers featuring specific restriction sites SunI/MunI flanking thegene and then inserted into SunI/MunI-digested pTHSN to createpTHSN-GMCSF (described in Cerullo et al. 2010, Cancer Res 70:4297-309).pAd5/3-D24-GMCSF was generated by homologous recombination in E. colibetween FspI-linearized pTHSN-GMCSF and Sill-linearized pAd5/3-D24(described in Kanerva et al. 2003, Mol Ther 3:449-58). The E2F-1promoter was amplified by PCR with specific primers with restrictionenzyme cutting sites for NotI and XhoI designed so that the promotercould be inserted into a pSE1.D24 plasmid (described in Nettelbeck etal. 2002, Cancer Res 62:4663-70 as pScsΔ24) to control E1A. Theresulting plasmid, pE2F.E1.D24 (SEQ ID NO: 8), contains the E2F-1promoter controlling E1A gene that has a 24 by deletion in CR2.pAd5/3-E2F-D24-GM-CSF (SEQ ID NO: 9) was generated by homologousrecombination in E. coli between PmeI-linearized pE2F.E1.D24 andSill-linearized rescue plasmid pAd5/3-D24-GMCSF (described in Koski etal. 2010, Mol Ther 18:1874-84). CGTG-602 virus genome was released byPacI digestion and transfection to A549 cells for amplification andrescue. All phases of the cloning were confirmed by multiple PCRs andrestriction digestions as well as sequencing for the relevant areas ofthe plasmids. All phases of the virus production, includingtransfection, were done on A549 cells to avoid risk of wild typerecombination. Other cancer cell lines known in the art that grow as aneven cell layer when cultured in vitro can also be used for producingthe virus.

Example 2 In vitro Analysis of CGTG-602 Virus

Functionality of the CGTG-602 produced GM-CSF was tested by analyzingthe proliferative activity of the GM-CSF dependent TF-1 erythroleukemiacells upon addition of filtered supernatant from CGTG-602 infected A549cells. GM-CSF dependent TF-1 erythroleukemia cells (Sigma Aldrich) werecultured in suspension in complete growth medium supplemented with 2 ngrecombinant hGM-CSF and kept on a shaker. A549 cells were grown ingrowth medium supplemented with 2% FCS and infected with 10 VP/cell ofAd5/3-E2F.D24-GM-CSF. 48 hours later the supernatant was collected andfiltered through a 0.02 μm inorganic filter (Whatman, Maidstone, UK). TF1 cells were centrifuged and resuspended into growth medium withouthGM-CSF, seeded on a 96-well plate at a density of 1×10⁴ cells/well andkept on a shaker. 0.1, 1 and 10 μl of filtered supernatant fromAd5/3-E2F.D24-GM-CSF infected A549 cells was added on TF 1 cells (6wells per each) and hGM-CSF was added on positive control cells. TF-1cells without supplementation were used as negative control. Three dayslater fresh growth medium without hGM-CSF was added on the cells. Cellviability was analyzed with a(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphopheny-1)-2H-tetrazolium) (MTS) cytotoxicity assay (Promega) after 5 days ofincubation as previously described (Koski et al. 2010, Mol Ther18:1874-84). The viability of positive control cells were depicted as100%.

In vitro efficacy of CGTG-602 virus was studied in lung cancer cells(A549), ovarian cancer cells (SKOV3.ipl) and prostate cancer cells(PC3-MM2) by utilizing MTS cell killing assays. MTS assay is currentlythe standard method to assess cell viability in cancer gene therapypublications. Ad5/3 Lucl is a replication deficient virus and acts as anegative control. Ad5 wt is a wild type Ad5 virus (strain Ad300 wt) andwas used as a positive control. Ad5/3-D24-GMCSF is an otherwise isogeniccontrol virus that has the native E1A promoter. VP indicates virusparticles. Cells were seeded on a 96-well plate at a density of 1×10⁴cells/well and infected after 24 hours with 1, 10 or 100 VP/cell.Infection was done in 50 μl of growth media supplemented with 2% FCS and1 hour later growth media with 10% FCS was added on cells. Thereafter,cells were maintained in 10% media and followed daily. MTS-assay wasperformed as previously described 6 days (A549 and PC3-MM2) or 14 days(SKOV3.ipl) later, when 100% cell killing with the highest viral dosewas observed. In summary, CGTG-602 had oncolytic activity similar topositive control virus Ad5/3-D24-GMCSF in vitro, and therefore theinsertion of E2F-1 promoter did not compromise the oncolytic potency ofthe virus (FIG. 3).

In vitro selectivity of CGTG-602 was analyzed by viral burst assay fromprimary human hepatocytes (Lonza). 24-well plate was coated with 0.5mg/ml Rat tail type 1 collagen in 1 mM acetic acid for 30 minutes.1.5×10⁵ primary human h NHEPS hepatocytes (Lonza) were seeded per wellin HCM medium with 2% FBS. Cells were let to rest for 3 hours in 37° C.and the growth media was changed to HCM without FBS. 24 hours later thehepatocytes were infected with 10 VP/cell of CGTG-602, CGTG102, Ad5 wtor growth media only. Growth media was changed after 2 hours ofinfection. Cells and supernatant were collected 24, 48 and 72 hoursafter infection and frozen at -80° C. Cells and supernatant went through4 cycles of freezing and thawing prior to the subsequent plaque assay.Standard plaque assay was performed with serially diluted (10⁻¹ to10⁻¹¹) supernatant from the samples centrifuged for 25 minutes at 4000rpm prior to infection. Each analysis contained a mock infected well forcomparison. In summary, CGTG-602 titers were 11-37-fold lower thanCGTG-102 titers and, at best, 64-fold lower than Ad5 wt titers (FIG. 4),representing enhanced selectivity of CGTG-602 in human primaryhepatocytes in vitro due to the E2F-1 promoter.

Example 5 In vivo Analysis of CGTG-602 Virus in Animals

The in vivo specificity of CGTG-602 was analyzed in immunocompetentSyrian hamsters, which are semipermissive for human adenovirusreplication (mice are non-permissive) (Ying B. et al. 2009, Cancer GeneTher doi:10.1038/cgt.2009.6.). Hamster HAPT-1 tumors were induced intothe flanks of the hamsters. After the tumors reached approximately thesize of 0.5 cm (7 days), 3×10⁸ VP of CGTG-602 was injectedintratumorally (n=8 tumors/timepoint). Hamsters without tumors(n=2/timepoint) were injected directly into the liver. Animals werekilled and tumors or livers collected 0.5, 24, 48, 72 or 96 hours aftervirus injection and stored at −80° C.

For quantitative PCR, frozen tissues were homogenized and total DNA wasextracted using the QIAamp Mini Kit (Qiagen, Valencia, Calif.) accordingto the manufacturer's instructions. Adenoviral E4 gene was used as thetarget for quantitative PCR and hamster glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used as an internal control target and tonormalize viral DNA copies per amount of genomic DNA. Quantitative PCRwas done using primers E4-forward (GGAGTGCGCCGAGACAAC, SEQ ID NO:10) andE4-reverse (ACTACGTCCGGCGTTCCA, SEQ ID NO:11) for E4, GAPDH-forward(CACCGAGGACCAGGTTGTCT, SEQ ID NO:13) and GAPDH-reverse(CATACCAGGAGATGAGCTTTACGA, SEQ ID NO:14) for GAPDH and probes E4-probe(6-FAM-TGGCATGACACTACGACCAACACGATCT-TAMRA, SEQ ID NO:12) for E4 andGAPDH-probe (6-FAM-CAAGAGTGACTCCCACTCTTCCACCTTTGA-TAM RA, SEQ ID NO:15)for GAPDH. A regression standard curve for GAPDH was established usingknown amounts of DNA extracted from cultured cells (1,800-0.18 ng).

In vivo efficacy of CGTG-602 was tested in immune competent Syrianhamsters, which are semipermissive for human adenovirus replication(mice are non-permissive) (Ying B. et al. 2009, Cancer Gene Therdoi:10.1038/cgt.2009.6.). 7×10⁶ HapT1 pancreatic cancer cells wereinjected subcutaneously into flanks, and when the tumors reached adiameter of approximately 0.5 cm, they were injected intratumorally with3×10⁸ VP of either CGTG-602 (n=6 hamsters, 24 tumors) or CGTG-102 (n=5hamsters, 20 tumors) 3 times every 3 days, and the tumor volumes werefollowed (FIG. 6). Further, 5 animals received 2 mg/hamstercyclophosphamide intraperitoneally in a combination with CGTG-602, 5hamsters received cyclophosphamide only and 4 animals were mock-treatedwith intratumoral growth medium injections. Treatments with CGTG-602slowed down the tumor progression significantly when compared to themock treatments. There was no significant difference between CGTG-602and CGTG-102 treatments in efficacy (although tumors in the former groupwere smaller), which is in line with the hypothesis that they areexpected to be as effective, but CGTG-602 has improved safetyproperties. Further, the combination treatment with cyclophosphamide didnot significantly enhance the efficacy of virus alone, even if meantumor size the combination group was slightly smaller. The HAPT-1 tumormodel is such an aggressive model that tumor growth in control groupslimited the length of the experiment. Therefore, the immunologicalbenefit of low-dose cyclophosphamide probably did not have enough timeto become visible. This is supported by the observation that tumors werebecoming smaller in the combination group at the last time point whiletumors were growing in all other groups. The model is not optimal forefficacy studied as hamster cells are likely to express only low levelsof desmoglein 2, which is the primary receptor for Ad3 based vectors.This is highlighted in other experiments, where CGTG-603, a virus withan Ad5 knob featuring an RGD modification, was used (FIGS. 7A-7C).

The receptors for this virus are known to be expressed in hamstertissues. 1×10⁹ VP of virus was administered on days 0, 2 and 4.Ad5-D24-RGD (lacks E2F-1 promoter and GM-CSF gene), Ad5-RGD-D24-GMCSF(lacks E2F-1 promoter) and Ad5 wt were used as control viruses. Mocktreated animals received growth medium only. All viruses eradicate thetumors within 16 days following the treatments (FIG. 7A). A similarsetup was used for the re-challenge experiment, where HAPT-1 tumors werefirst allowed to grow and were treated as mentioned above (FIG. 7 B),and subsequently were surgically removed. The same hamsters werere-challenged with HAPT-1 cells and treated as above. CGTG-603 treatmentresulted in complete protection against tumor re-challenge, highlightingthe immunostimulatory role of the transgnene GM-CSF (FIG.

7C).

Example 6 Analysis of CGTG-602 in Human Patients I. Patients

Patients with advanced and treatment refractory solid tumors wereenrolled in a FIMEA regulated Advanced Therapy Access Program treatmentprotocol (ISRCTN 10141600, EC/1394/2007). Information of patientsreceiving CGTG-602 is listed in Table 1.

14 patients with advanced solid tumors refractory to standard therapies(Table 1) were serially treated with CGTG-602 intravenously andintratumorally (Table 2). Intratumoral injection was performedintraperitoneally or intrapleurally in the case of carcinomatosis orpleural metastases, respectively. Inclusion criteria were solid tumorsrefractory to conventional therapies, WHO performance score 2 or lessand no major organ function deficiencies. Exclusion criteria were organtransplant, HIV, severe cardiovascular, metabolic or pulmonary diseaseor other symptoms, findings or diseases preventing oncolytic virustreatment. Written informed consent was obtained and treatments wereadministered according to Good Clinical Practice and the Declaration ofHelsinki.

II. Treatments with Adenoviral Vector Encoding GM-CSF

a) CGTG-602 Treatments

13/14 patients were serially treated with CGTG-602 for 2-4 timesapproximately every 3 weeks. One patient received 1 treatment ofCGTG-602 (C332) and subsequently serial treatment with another virus.This patient is included only for an immunohistochemical analysis ofCD8+ T-cells from a tumor sample that was obtained 3 weeks aftertreatment with CGTG-602 and for assessment of adverse events after thesingle treatment, and is not included in any of the responseevaluations.

Virus administration was performed by ultrasound-guided intratumoralinjection and circa one fifth of the dose was given intravenously in thefirst injection. In subsequent injections the entire dose was givenintratumorally. Viral doses ranging from 3×10¹° VP to a maximum of1×10¹² VP were used based on safety results previously published withAds/3-D24-GM-CSF (Koski et al. 2010, Mol Ther 18:1874-84).

Virus was diluted in sterile saline solution at the time ofadministration under appropriate condition. Following virusadministration all patients were monitored overnight at the hospital andsubsequently during the whole treatment period and after the lasttreatment for 4 weeks as outpatients. Physical assessment and medicalhistory were done at each visit and clinically relevant laboratoryvalues were followed. Side effects of treatment were recorded and scoredaccording to Common Terminology for Adverse Events v3.0 (CTCAE).

Because many cancer patients have symptoms due to disease, pre-existingsymptoms were not scored if they did not become worse. However, if thesymptom became more severe, e.g. pre-treatment grade 1 changed to grade2 after treatment, it was scored as grade 2. Tumor size was assessed bypositron emission tomography-contrast-enhanced computer tomography(PET-CT) scanning. A modification of the PET Response Criteria in SolidTumors 30 were applied to overall disease, including injected andnon-injected lesions. No adjustments for fat % or body surface area wereperformed. The five most active lesions, maximum two lesions per organ,were evaluated for SUVmax and the values were summed. Lymph node signalincrease was not considered progression as lymph node metabolismincreases during inflammation and eg. after vaccination. Progressivemetabolic disease (PMD)=30% increase of Summed SUVmax or >2 cm PETpositive new lesion. Stable metabolic disease (SMD)=−9%−+29% change,minor metabolic response (MMR)=−29%−10% decline, partial metabolicresponse (PMR)=−30% decline in Summed SUVmax. Complete metabolicresponse (CMR)=disappearance of all metabolically active tumor. Tumormarkers were measured from serum when elevated at baseline, and the samepercentages were used.

Table 3 reports the efficacy evaluation of CGTG-602 according to thecriteria described above.

b) Safety of CGTG-602 in Cancer Patients

Treatments were well tolerated up to the highest dose used: 1×10¹²VP/patient. Table 4 summarizes all the adverse events that were recordedduring CGTG-602 treatment rounds. Adverse events are reported asfrequency per all 39 treatment rounds. All the adverse events have beengraded according to Common Terminology for Adverse Events v3.0 (CTCAE).No grade 4-5 adverse events were seen. Fever (22/39 treatment rounds),fatigue (22/39 treatment rounds) or upper respiratory symptoms (7/39treatment rounds) were common grade 1-2 flu-like symptoms. AST elevation(11/39), pain in the injection site (6/39 treatment rounds), abdominalpain (15/39 treatment rounds), nausea (11/39 treatment rounds), vomiting(7 treatment rounds) and oedema (6/39 treatment rounds) were alsorelatively common grade 1-2 adverse events. Grade 3 symptoms were seenin 11/39 treatment rounds: fever (2/39), neuropathy (1/39), pain (5/39),upper respiratory symptoms (2/39), oedema (1/39) and flushing (1/39).Asymptomatic and self-limiting grade 3 hematological or metabolic sideeffects were seen in 6/39 treatment rounds: anemia (2/39), hyponatremia(2/39), leucosytopenia (1/39) and creatinine elevation (1/39).

III. Virus Replication

Serum samples were collected from patients treated with CGTG-602 andconventional PCR was carried out with primers and conditions accordingto Takayama et al. 2007, J. Med. Virol. 79:278-284. Briefly, total DNAwas extracted by adding 3 pg of carrier DNA (polydeoxyadenylic acid;Roche, Mannheim, Germany) to 400 μl of serum and using the QIAamp DNAmini kit. Extracted DNA was eluted in 60 μl nuclease-free water and DNAconcentration was measured by spectrophotometry. PCR amplification wasbased on primers and probe targeting the E1A region flanking the 24-bpdeletion (E1 forward primer 5″-TCCGGTTTCTATGCCAAACCT-3 (SEQ ID NO:16),E1 reverse primer 5′-TCCTCCGGTGATAATGACAAGA-3′ (SEQ ID NO:17) and probeonco 5′^(FAM)-TGATCGATCCACCCAGTGA-3′^(MGBNFQ) (SEQ ID NO:18)). Inaddition, a probe complementary to a sequence included in the 24-bpregion targeted for deletion was used to test the samples for thepresence of wild-type adenovirus infection (probe wt5′^(VIC)-tacctgccacgaggct-3′^(MGBNFQ) (SEQ ID NO:19)).

The real-time PCR conditions for each 25 μl reaction were as follows: 2×LightCycler480 Probes Master Mix (Roche, Mannheim, Germany), 800 nM eachforward and reverse primer, 200 nM each probe and 250 ng extracted DNA.PCR reactions were carried out in a LightCycler (Roche, Mannheim,Germany) under the following cycling conditions: 10 min at 95° C., 50cycles of 10 s at 95° C., 30 s at 62° C. and 20 sec at 72° C. and 10 minat 40° C. All samples were tested in duplicate. TaqMan exogenousinternal positive control reagents (Applied Biosystems) were used in thesame PCR runs to test each sample for the presence of PCR inhibitors.

A regression standard curve was generated using DNA extracted fromserial dilutions of Ad5/3-D24-Cox2L (1×10⁸⁻¹⁰ vp/ml) in normal humanserum. The limit of detection and limit of quantification for the assaywere 500 vp/ml of serum.

Positive samples were confirmed by real-time PCR using LightCycler480SYBR Green I Master mix (Roche, Mannheim, Germany) and primers specificfor adenovirus and GM-CSF sequences (GM-CSF forward primer5″-AAACACCACCCTCCTTACCTG-3′ (SEQ ID NO:20) and GM-CSF reverse primer5″-TCATTCATCTCAGCAGCAGTG-3′ (SEQ ID NO:21)).

All patients evaluated for the presence of CGTG-602 in serum werenegative for CGTG-602 prior to the treatment (Table 3). On day 1 afterthe first treatment September 11 evaluable patients had measurablelevels of virus genomes in the serum, with the highest titer being 1141VP/ml serum. From samples taken during days 3-7 2/4 evaluable patientswere positive, with the highest titer of 11523 VP/ml serum, suggestingvirus replication at tumors.

III. Detection of CD8+ T-Cells from Tumor

Tumor sample from patient C332 was obtained 4 weeks after treatment withCGTG-602. Tumor and normal peritoneal lining were fixed in 4% formalinand paraffin blocks were made. For analysis of CD8 positive cells, i.e.cytotoxic T-cells, tissue sections of 4 μm thickness were prepared,deparaffinized, rehydrated and incubated with a primary mouse anti-CD8antibody (NCL-CD8-4B 11; Novocastra, Newcastle Upon Tyne, UnitedKingdom) at a dilution of 1:25 in antibody diluent 50809(DakoCytomation, Carpinteria, Calif., USA). Sections were washed andincubated with a secondary anti-mouse antibody labeled with horseradishperoxide (HRP) and counterstained for hematoxyline. Pictures were takenwith an Axioplan2 microscope (Carl Zeiss) equipped with Axiocam (Zeiss).Infiltration of CD8+ T-cells was seen in tumor samples but not in normalperitoneal lining, suggesting anti-tumor immunity induction by CGTG-602(FIGS. 8A and 8B).

IV. Efficacy of CGTG-602

All patients had progressing tumors prior to treatment. 7 patients couldbe assessed for radiological benefit according to PERCIST (Table 3). Ofthe 6 evaluable patients, 1 patient had a complete response (CMR), 1patient had partial response (PMR) and a complete response in anon-injected mediastinal lesion, 1 patient had minor response (MMR), 2patients had a stable disease (SMD) and 1 patient had progressivedisease (PMD). Therefore, the radiological disease control rate was 83%of the 6 radiologically evaluable patients while the response rate(including MMR) was 50%.

Patient R319 had a 49.1% reduction in metabolic activity of an injectedliver lesion (FIGS. 13C-13D) and a non-injected mediastinal lesiondisappeared (FIGS. 13A-13B) after treatment.

A complete metabolic response was seen in PETCT imaging tumor of thetumor in the right lung of patient 5354 (FIGS. 14A-14F): (FIG. 14A)baseline, (FIG. 14B) after 3 months of treatment, (FIG. 14C) after 6months, (FIG. 14D) after 9 months. Tumor size started diminishing at 6months. (FIGS. 14E-14F) a different plane of PET analysis from the samepatient at baseline and after 3 months, arrows indicate PET activeregions of the tumor.

With regard to tumor markers, assessed for patients who had elevatedmarkers at baseline, 3/9 patients had reduction of marker levels, 2/9had initial reduction and subsequent elevation of marker levels, 1patient had initial elevation and subsequent reduction and 3/9 hadelevation of marker levels (FIG. 11).

Overall, with regard to tumor marker or radiological responses, signs ofantitumor efficacy were seen in 9/12 evaluable patients (75%). Thesepatients lived a median of 135 days while the median survival of theother three was 80 days. Overall survival of all patients discussed hereis shown in FIG. 12.

V. Immune Responses Elicited by the Treatment

a. T-Cell Responses

Oncolytic cell death allows the immune system to gain the capacity forrecognizing and killing tumor cells. This is potentially beneficial fortumor eradication and may facilitate cures. Adenovirus is cleared outfrom the body in a relatively short time following the administration;hence it becomes of key importance to stimulate the immune system to beable to recognize specific tumor-antigen so that the treatment canresult in a sustained beneficial effect for the patient. In addition, inthe presence of antibody, the virus is neutralized so that it can loseits efficacy of infecting metastasis. However, effector T or NK cellsinduced against the tumor are free to circulate and eventually killmetastasis far from the injected tumor. In order to demonstrate that theGMCSF-expressing adenovirus is able to elicit adenovirus- and tumorspecific immunity, PBMCs collected from treated patients were analyzedby IFN-gamma ELISPOT (IFN-gamma is a specific activation marker ofstimulated T cells). In FIG. 9A are illustrated the results from suchanalysis. The frequency of IFN-gamma producing tumor associated antigen(TAA) (survivin alone, CEA+NY-ESO-1 or cmyc+SSX2) specific PBMCsincreased 6/13 patients and decreased in 4/13 patients after treatment(FIG. 9A). The frequency of adenovirus specific (penton) IFN-gammaproducing PBMCs increased in 9/13 patients after treatment. Theseresults suggest that the PBMC-population gained “anti-tumor”characteristics. Further, reduction of the frequency of stimulated PBMCsin the blood may suggest trafficking from the periphery to the tumorsite.

Interestingly, we discovered that there was a 75% concordance ininduction of antiviral and antitumor cells on a patient level (FIG. 9A).In other words, if there was an increase in anti-viral cells, most ofthose patients also featured an increase in one or more classes ofanti-tumor T-cells. Unexpectedly, this finding reveals that in humans,anti-viral response corresponds and may contribute to anti-tumorresponse through epitope spreading and reduction of tumor-associatedimmunological tolerance. The concordance in induction of antiviral andantitumor cells was 100% when the antigen specific PBMCs was viewedproportionate to all IFN-gamma producing PBMCS (FIG. 9B).

b. Antibody Responses to Tumor Antigens

Antibodies against tumor associated antigens (TAA) are often elevated incancer patients. Serum samples from patients treated with CGTG-602 wereanalyzed with indirect

ELISA for antibodies against NY-ESO-1 MUC-1 (Ca15-3), CEA and survivin.Shortly, 200 μl of proteins (NY-ESO-1 and CEA) or peptides (survivin andMUC-1) in a concentration of 0.5 μg/ml were added on immulon 2HB plates(Thermo scientific, Milford, Mass., USA) for overnight coating at 4° C.Free binding sites were blocked with 2.5% BSA at room temperature. Serumsamples were diluted 1:100 and added on the wells incubated at roomtemperature for 2 hours. After washing, plates were incubated withanti-human IgG conjugated to alkaline phosphatase and after washing, 1step PNPP substrate (Pierce) was added for appropriate color reaction.After incubation for 30 minutes, reaction was stopped with 2M NaOH andabsorbance at 405 nm was read. As a control, serum from 5 healthy donorswas used to establish a cutoff value for elevated antibody level. Cutoffvalue was determined as the mean absorbance of the normal samples plus 2standard deviations.

The changes in the antibody levels of those patients that had anelevated level at any timepoint are presented as percentage of thepre-treatment level in FIG. 10. Frequently, the elevated antibody levelsdecreased to normal levels due to the treatment with patients thatotherwise benefited from the treatment (i.e. had decrease in elevatedtumor marker levels or radiologic response). On the contrary, two of thepatients that did not benefit from the treatment showed clear elevationin the antibody levels despite treatment (represented as black bars inFIG. 10). To this end, it was previously shown that a decrease inNY-ESO-1 antibody titers are associated with tumor regression (Jager eta. 1999, Int J Cancer 84:506-510). Furthermore, decrease of antibodylevels in serum may indicate trafficking to tumor site.

Example 8 The Effect of KKTK Mutation on Viral Transduction

To study the effect of the mutation on KKTK motif on viral transductionto cancer and liver, viral load was analyzed in liver and tumors ofintravenously treated mice. Briefly, 2×10⁶ M4A4-LM3 cells wereinoculated to both upper most mammary fat pads of Nude NMRI mice andtumors were let to develop until circa 5 mm in diameters. 5×10¹⁰ VP ofAd5/3 luc*(KKTK mutated virus) or the control virus Ad5/3 lucl or Ad5lucl were administered by a single intravenous injection to the tailvein. After 30 minutes mice were sacrificed and livers and tumorscollected. Viral load in the tissues was quantified by qPCR with primersand probes against adenoviral e4 region. qPCR for mouse 3-actin (primermouse beta-actin-forward: CGACGCGTTCCGATGC, SEQ ID NO:25; primermouse-beta-actin-reverse: TGGATGCCACAGGATTCCAT, SEQ ID NO:26; probemouse beta-actin: ^(6FAM)-AGGCTCTTTTCCAGCCTTCCTTCTTGG-TAMRA, SEQ IDNO:27) and human (3-actin (primer Human beta-actin-forward:CAGCAGATGTGGATCAGCAAG, SEQ ID NO:22; primer Human beta-actin-reverse:CTAGAAGCATTTGCGGTGGAC, SEQ ID NO:23; probe Human beta-actin:^(6FAM)-AGGAGTATGACGAAGGCCCCTC-TAMRA, SEQ ID NO:24), for liver and tumortissues respectively, was used to normalize viral titers to tissue DNA.The results indicate that the KKTK mutated Ad5/3 luc*exhibits reducedliver transduction while retaining tumor transduction in vivo (FIG. 15).

To further verify that the KKTK mutation does not hinder viraltransduction to cancer cells, in vitro transduction to various cancercell lines was assessed (FIG. 16). Hey (ovarian adenocarcinoma), PC-3(prostate cancer), Skov3.ipl (ovarian adenocarcinoma), and M4A4-LM3(breast ductal carcinoma) were infected with 200 VP/cell. Unbound viruswas washed out after 1 h incubation and regular growth media was added.Cells were lysed and frozen after a total of 24 hours of incubation.Luciferase transgene activity was quantified as relative light units(RLU) by measuring luminosity emitted from cell lysates after additionof luciferin, the substrate for luciferase transgene. Capped barindicates standard deviation of mean. The results show that the mutationin the fiber KKTK motif does not hamper viral transduction to cancercell lines in vitro.

Example 9 The Effect of CpG

To analyze the feasibility of CpG islands in human cells, NFkBactivation by pTHSN plasmid containing either one or two CpG islands wasanalyzed in 293-hTLR9 (Invivogen) cell line. 293hTLR9 is a cell linethat expresses exclusively TLR9. Cells were transfected with a plasmidexpressing luciferase driven by an NFkB-inducible promoter. 24 hourslater pTSHN, pTSHN-CpG1 (with 1 island) and pTSHN-CpG2 (with 2 islands)were added to the media and 12 hours later luciferase was measured (FIG.17). The luciferase activity was dependent on NFkB activation. Bindingof CpG to TLR9 causes a conformational shift in the receptor, causingactivation of NFkB. pTHSN without CpG was used as a negative controlplasmid, lipopolysaccharide (LPS), a frequently used stimulator ofsignaling pathways and NFkB was used as a positive control andluciferase signal from mock treated cells were subtracted as background.The results indicate that the insertion of CpG to the viral genomefunctions through TLR9 to activate NFkB of the responding cell.

Example 10 Statistical Analysis

Two-tailed student's T-test was used to analyze the in vitro efficacy aswell as the infective virus load in hepatocytes. One way analysis ofvariance (ANOVA) was used to assess tumor volume for hamsterexperiments. Survival data was processed with Kaplan-Meier analysis.

TABLE 1 Patients at baseline Patient Age ID (y) Sex Diagnosis Priortherapies WHO O314 62 F Ovarian cancer surgery, docetaxel + carboplatin(×2), doxorubicin, weekly-paclitaxel, carboplatin, gemcitabine, 1topotecan O337 69 F Ovarian cancer surgery × 2, carboplatin +paclitaxel, carboplatin (×2), radiotherapy, carboplatin, topotecan, 2doxorubicin, tamoxifen O340 74 F Ovarian cancer surgery (×5), bleomycin,etoposide, cisplatin, letrozole 0 O351 72 F Ovarian cancer surgery,paclitaxel + carboplatin (×3), gemcitabine + carboplatin, gemcitabine,doxorubicin, 2 topotecan, etoposide, oxaliplatin, vinorelbin, tamoxifenC312 54 M Rectum cancer capecitabine, capecitabine + oxaliplatin,capecitabine + oxaliplatin + bevacizumab, irinotecan + 1 bevacizumab,bevacizumab, cetuximab + 5-FU − irinotecan (×2), oxaliplatin + 5FU,bevacizumab C332* 49 F Colon cancer surgery × 2, capecitabine +oxaliplatin + bevacizumab, capecitabine + irinotecan + bevacizumab, 0capecitabine + bevacizumab H192 54 M Pancreatic cancer gemcitabine,capecitabine, gemcitabine chemoradiation, gemcitabine + erlotinib 1 H34458 F Pancreatic cancer surgery, gemcitabine + erlotinib, gemcitabine +capesitabine, gemcitabine + oxaliplatin + 1 capesitabine, gemcitabine +erlotinib I347 51 M Melanoma surgery, DTIC + interferon, paclitaxel +carboplatin 2 R319 67 F Breast cancer docetaxel (multiple), gemcitabine,cyclophosphamide + epirubicin + fluorouracil, capecitabine, 1vinorelbine + epirubicin + 5-FU, doxorubicin, tomerifene, letrozole,fulvestrant, exemestane, medroxyprogesterone acetate R342 54 F Breastcancer surgery × 2, cyclophosphamide, epirubicin, 5-FU, radiotherapy,docetaxel + capecitabine, 2 capecitabine, doxorubicin, cisplatin +gemcitabine R356 40 F Breast cancer surgery × 4, radiotherapy × 2,cyclophosphamide + epirubicin + 5-FU, vinorelbine + trastuzumab, 1capecitabine + lapatinib, paclitaxel + bevacizumab, docetaxel +epirubicin + capecitabine + bevacizumab, paclitaxel + gemcitabine S35259 F Sarcoma surgery × 2 1 S354 50 F Fibrosarcoma surgery, ifosfamide +doxorubicin (×6), radiotherapy 2 *patient C332 received only 1 CGTG-602treatment prior to other virus treatments and is not included in allanalysis

TABLE 2 Treatment dose, number of treatments and virus sensitizersPatient Treatment dose, Virus ID no. of treatments sensitizers O314 1 ×10¹¹ (1) cyclophosphamide 3 × 10¹¹ (2) O337 5 × 10¹¹ (3)cyclophosphamide O340 5 × 10¹¹ (3) cyclophosphamide O351 3 × 10¹¹ (3)cyclophosphamide C312 1 × 10¹¹ (1) cyclophosphamide 3 × 10¹¹ (2) C332 8× 10¹¹ (1) — H192 3 × 10¹¹ (2) cyclophosphamide, temozolomide, erlotinibH344 8 × 10¹¹ (3) cyclophosphamide, temozolomide I347 5 × 10¹¹ (3)cyclophosphamide, temozolomide R319 3 × 10¹¹ (1) cyclophosphamide 5 ×10¹¹ (2) R342 3 × 10¹¹ (2) cyclophosphamide R356 1 × 10¹² (4)cyclophosphamide S352 3 × 10¹¹ (1) cyclophosphamide, 1 × 10¹² (2)temozolomide S354 3 × 10¹¹ (4) cyclophosphamide, temozolomide

TABLE 3 Virus load in serum Days post treatment Treatment responsesPatient after 1^(st) after 2nd after 3rd PET or Survival ID 0 1 3-8 0 13-8 0 1 3-10 14-55 (%) Marker Other benefit (days) O314 0 0 0 <500 0 0mPR  71 O337 0 <500 0 0 <500 mSD 273*  O340 0 1141 3608 0 <500 0 0 <5000 0 MMR mCR 258*  (−10.3%) O351 0 0 <500 0 0 0 0 mMR  87 C312 0 <500 0<500 <500 SMD mPD 349* (+23%) H192 0 0 0 <500 mPD  80 H344 0 <500 115230 <500 0 0 <500 0 0 PMD mPD 133 I347 0 <500 0 0 <500 0 0 <500 106 R319 00 0 0 0 0 <500 PMR mPR CR: non-injected 332 (−49.1%) mediastinal lesionR342 0 <500 <500 mPD  76 R356 0 <500 <500 876 <500 mPR 102 S352 0 0 0<500 0 0 0 0 SMD 112 (+6.1%) S354 0 789 0 0 <500 0 CMR 39% reduction intumor 135*  (−39%)^(a) volume symptoms: clear improvement blanksindicate sample not available *patient still alive MMR: minor metabolicresponse; PMR: partial metabolic response; SMD; stable metabolicdisease; PMD: progressive metabolic disease; CMR: complete metabolicresponse; mMR: marker minor response; mPR: marker partial response; mSD:marker stable disease; mPD: marker progressive disease; mCR: markercomplete response

TABLE 4 Adverse events (occurring in percentage of 39 rounds of therapyin 14 patients) Grade 1 Grade 2 Grade 3 Grade 4-5 Constitutional Fever28 28 5 — Fatigue 10 46 — — Chills 8 3 — — Rigors 8 3 — —astrointestinal norexia 5 10 — — ausea 13 15 — — omiting 13 5 — —Heartburn 8 — — — Diarrhea 3 5 — — Constipation 5 3 — — Distension 5 3 —— Ileus — 3 — — Feel of satietas — 3 — — eurologic and Ocular europathy5 — 3 — Muscle cramps 3 — — — Pain In ection site 13 3 — — bdominal 1821 3 — imbs 3 13 — — Back — 5 — — Chest wall 3 — 3 — Headache 8 3 — —Flank — 5 3 — Others 18 15 5 — Pulmonary Upper respiratory Pneumothorax3 — — — Dyspnea 5 5 3 — Cough 3 3 — — ung infection — — 3 —Hematological nemia 13 26 5 — eukocytopenia 10 8 3 — Thrombocytopenia 18— — — Metabolic aboratory ST elevation 26 3 — — Hypokalemia 3 — — —Hyponatremia 15 — 5 — Creatinine elevation — — 3 — I R elevation 3 — — —Other Oedema 8 8 3 — Urine incontinence — 3 — — Hemorrhage 5 — — —Flu-like symptoms 3 5 — — Flushing — — 3 — lopecia 3 — — — Pruritus — 3— — oice changes 3 — — —

What is claimed is:
 1. A recombinant serotype 5 (Ad5) adenoviruscomprising: (a) a nucleic acid sequence encoding the wild-type EIAadenoviral promoter or encoding a E2F-1 promoter replacing the wild-typeEIA adenoviral promoter; (b) a 24 by deletion (D24) in the Rb bindingconstant region 2 of E1; (c) a deletion in gp19k/6.7K in E3; (d) anucleic acid sequence encoding a human CD40 ligand in the deletion ingp19k/6.7K, the nucleic acid sequence under the control of the E3promoter; and (e) a capsid modification wherein a region encoding Ad5adenoviral fiber knob is replaced by the corresponding region from aserotype 3 (Ad3) adenovirus.
 2. The recombinant Ad5 adenovirus accordingto claim 1, wherein the nucleic acid sequence encodes the wild-type EIAadenoviral promoter.
 3. The recombinant Ad5 adenovirus according toclaim 1, further comprising at least one element selected from the groupconsisting of an adenoviral immediate early gene, an adenoviralintermediate gene, and an adenoviral late gene.
 4. A method of treatinga cancer comprising administering to a subject in need thereof aneffective amount of the recombinant Ad5 adenovirus according to claim 1.5. The method of claim 4, wherein a first administration of therecombinant Ad5 adenovirus to the subject is followed by a subsequentadministration or several administrations.
 6. The method of claim 5,wherein the subsequent administration comprises a different recombinantAd5 adenovirus from the recombinant Ad5 adenovirus administered in thefirst administration.
 7. The method of claim 4 comprising a firstadministration or several administrations of the recombinant Ad5serotype adenovirus and a radiotherapy, a surgery, or administration ofone or more agents selected from the group consisting of a virussensitizer, a chemotherapeutic agent, verapamil, a calcium channelblocker, an anti-CD20 therapy, and an autophagy-inducing agent.
 8. Themethod of claim 4, wherein the recombinant Ad5 adenovirus is capable ofreplicating and having lytic activity in a target cell and wherein therecombinant Ad5 adenovirus is unable to bind Rb, thereby preventingvirus replication outside a target cell.
 9. The method of claim 4,wherein the deletion in gp19k/6.7K in E3 disrupts the recombinant Ad5adenovirus' ability to control a host immune response.
 10. A method ofproducing and recovering infectious recombinant Ad5 adenovirus particlescomprising steps of: (a) providing a recombinant Ad5 adenovirusaccording to claim 1 inside a host cell permissive for adenovirusreplication, (b) culturing the host cell under conditions that allowsaid recombinant Ad5 adenovirus to propagate and to produce infectiousrecombinant Ad5 adenovirus particles, and (c) recovering said infectiousrecombinant Ad5 adenovirus particles.
 11. A pharmaceutical compositioncomprising the recombinant Ad5 adenovirus according to claim 1 and apharmaceutically acceptable carrier.
 12. The pharmaceutical compositionof claim 11 formulated to contain 5×10¹⁰ to 5×10¹¹ viral particles. 13.A virus particle comprising the recombinant Ad5 adenovirus according toclaim
 1. 14. A host cell comprising the recombinant Ad5 adenovirusaccording to claim
 1. 15. A method for inducing immunity directedagainst a cancer cell, for treating a tumor, or for preventing a tumor'sgrowth in a subject in need thereof comprising administering to thesubject an effective amount of the recombinant Ad5 adenovirus accordingto claim 1.